6.05A: Enzyme Reaction Mechanisms - Arrow Pushing

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Learning Goals (ChatGPT o1, 1/30/25)

1. Understand the Fundamentals of Arrow Pushing in Enzyme Mechanisms:

   • Define arrow pushing and its significance in illustrating electron flow during enzyme-catalyzed reactions.
   • Explain how arrow pushing provides insight into reaction mechanisms and intermediate states.

2. Analyze Mechanistic Steps in Enzymatic Reactions:

   • Identify and interpret key mechanistic steps in enzyme-catalyzed reactions using arrow pushing diagrams.
   • Evaluate the role of catalytic residues and cofactors in facilitating chemical transformations.

3. Apply Arrow Pushing Techniques to Reaction Mechanisms:

   • Develop the ability to construct and annotate arrow pushing diagrams for complex enzymatic reactions.
   • Practice using arrow pushing to predict reaction outcomes and intermediate species.

4. Critically Evaluate Mechanism Diagrams:

   • Assess the clarity and accuracy of enzyme reaction mechanisms depicted through arrow pushing.
   • Compare different mechanistic representations to determine the most plausible reaction pathway.

5. Integrate Biochemical Concepts with Mechanistic Analysis:

   • Connect the principles of enzyme structure and function with the detailed steps illustrated in arrow pushing diagrams.
   • Apply theoretical knowledge to real-world biochemical problems by analyzing enzyme reaction mechanisms.

6. Develop Problem-Solving Skills in Enzymology:

   • Enhance critical thinking by troubleshooting common mistakes and misconceptions in arrow pushing.
   • Use mechanistic insights to propose modifications or improvements in enzyme function based on structural changes.

prompt:  Write a series of learning goals for the following web page. The page is designed for junior and senior biochemistry majors.

 

KristenProcko and HenryJakubowski have written this chapter section.

We can apply what we learned about catalysis by small molecules (e.g., acids and bases) to enzyme-catalyzed reactions. To understand the mechanism of an enzyme-catalyzed reaction, we try to alter as many variables, one at a time, and ascertain the effects of the changes on the activity of the enzyme. Kinetic methods can be used to obtain data, from which inferences about the mechanism can be made. Crystal structures of the enzyme in the presence and absence of a competitive inhibitor give abundant information about possible mechanisms. It is amazing, however, how much information about enzyme mechanism can be gained even if all you have is a blender, a stopwatch, an impure enzyme, and a few substrates and inhibiting reagents.

Introduction

Almost every chemical reaction in the biological world is catalyzed by protein enzymes. The human genome encodes over 20,000 proteins, thousands of which are enzymes. The total number of different enzymes in the biosphere must be staggering. Yet simultaneously, all of these enzymes catalyze different sets of similar reactions. To bring order to the world of enzyme catalysis, the IUBMB has classified enzymes based on the type of chemical reactions they catalyze. There are seven main categories, as shown in the expandable Table \(\PageIndex{1}\) below. Each reaction type is given a four-digit Enzyme Commission number. For example, alcohol dehydrogenase, the enzyme that catalyzes the oxidation of ethanol, a primary alcohol, to acetaldehyde using an oxidizing agent called NAD⁺, is given the enzyme commission number EC 1.1.1.1. Other enzymes that oxidize primary alcohols to aldehydes or secondary alcohols to ketones are also given the same EC number.

Class	Subclass	Type	Description
EC 1	[+]	Oxidoreductases	redox reactions
EC 2	[+]	Transferases	transfer/exchange of group from one molecule to another
EC 3	[+]	Hydrolases	hydrolysis reactions
EC 4	[+]	Lyases	elimination forming a double bond
EC 5	[+]	Isomerases	conversions of geometric, stereo- or constitutive isomers
EC 6	[+]	Ligases	condensation of two molecules into one
EC 7	[+]	Translocases	movement of species across a semipermeable membrane

Table \(\PageIndex{1}\): ExplorEnz database for the curation and dissemination of the International Union of Biochemistry and Molecular Biology (IUBMB) Enzyme Nomenclature. (Source: https://academic.oup.com/nar/article...1/D593/1000297)

Enzymes with the same or similar EC numbers probably have similar reaction mechanisms. Throughout this book, we will explore the reaction mechanisms of many enzymes, but we can't and shouldn't explore all of them. You can take your acquired understanding of the reaction mechanism for key representative enzymes and apply it to others. Of course, experimental evidence is needed to validate a given mechanism. This chapter section will focus on the mechanisms of a few transferases (EC2) and hydrolases (EC3) as prototypical examples.

The rules for electron pushing in biochemical mechanisms mirror those from organic chemistry. However, because of the length of some biochemical mechanisms, abbreviated mechanisms are often accepted and will be presented in the literature and textbooks. This section aims to review arrow pushing by presenting some simple biochemical mechanisms and to familiarize you with acceptable alternative ways to show arrow pushing.

Class I Methyltransferases

Coenzymes are organic molecules that participate in some enzyme-catalyzed reactions (see Section 6.8 for a detailed discussion). These "enzyme helpers" often impart reactivity that an enzyme would not have. We'll begin our investigation with a mechanism catalyzed by class I methyltransferases using the coenzyme S-adenosylmethionine (SAM). SAM is formed from the reaction of methionine with ATP, resulting in the positively charged sulfur shown in Figure \(\PageIndex{1}\). The blue methyl group attached to the sulfur is very electrophilic due to the sulfur cation and is transferred to a nucleophilic substrate.

The reaction in Figure \(\PageIndex{1}\) shows the SAM-promoted conversion of norepinephrine to epinephrine. It is catalyzed by the enzyme phenylethanolamine N-methyltransferase (EC: 2.1.1.28). The nucleophilic nitrogen atom in norepinephrine is brought closer to the electrophilic methyl group when it binds to the enzyme. In an S_N2 reaction, nitrogen attacks, and we show a second arrow to keep track of the electrons from the carbon-sulfur bond, becoming a lone pair on the sulfur atom.

An amine has a pK_a close to 30, but a protonated amine has a pK_a around 8–10. Specifically, the conjugate acid of epinephrine has a pK_a near 8. Therefore, a protonated amine is a reasonable product to show at physiological pH (near 7.4) following the attack step. However, biochemical products are often shown uncharged when depicting an overall reaction. Therefore, a general base can be shown deprotonating the ammonium ion. The general base could be an amino acid side chain or a general base within the buffer components contained in a cell; we will represent the general base as B: here.

Figure \(\PageIndex{1}\): Class I methyltransferase mechanism with substrate norepinephrine

To simplify the mechanism in Figure \(\PageIndex{1}\), biochemists may abbreviate the arrow-pushing steps. The one-step deprotonation in Figure \(\PageIndex{2}\) shows that the general base deprotates the amine. The electrons from that bond are used to attack, and deprotonated epinephrine is produced in a single step with that arrow pushing. It is important to remember that anionic nitrogen is not made in the reaction mechanism. With pK_a values near 30, most amines are only deprotonated in practice using very strong organic bases, such as butyllithiums.

Because of this, the authors prefer the alternative arrow-pushing mechanism shown at the bottom of Figure \(\PageIndex{2}\). Partial bond formation between the nitrogen and the methyl group must occur before a biological general base can deprotonate norepinephrine, so showing the arrow coming from the lone pair on nitrogen is perhaps a better depiction of the biological reality. The arrow pushing shown in each methyltransferase mechanism is acceptable, and you may see each in different contexts.

Figure \(\PageIndex{2}\): Class I methyltransferase mechanism alternative arrow pushing

Kinases

Transfer of a phosphate group is common in biochemistry. The phosphate group is often transferred via kinase enzymes from adenosine triphosphate (ATP). The following excerpt from Chemistry LibreTexts describes the phosphate transfer mechanism:

In a phosphate transfer reaction, a phosphate group is transferred from a phosphate group donor molecule to a phosphate group acceptor molecule, as shown in Figure \(\PageIndex{3}\).

Figure \(\PageIndex{3}\): Phosphate transfer to an acceptor

A very important aspect of biological phosphate transfer reactions is that the electrophilicity of the phosphorus atom is usually enhanced by the Lewis acid (electron-accepting) effect of one or more magnesium ions. Phosphate transfer enzymes generally contain a Mg²⁺ ion bound in the active site in a position where it can interact with non-bridging phosphate oxygens on the substrate (Figure \(\PageIndex{4}\)). The magnesium ion pulls electron density away from the phosphorus atom, making it more electrophilic.

Figure \(\PageIndex{4}\): Phosphate interaction with magnesium ion

Without this metal ion interaction, phosphate is a poor electrophile, as the negatively charged oxygens shield the phosphorus center from attack by a nucleophile.

Note: For simplicity and clarity, we may omit the magnesium ion or other active site groups interacting with phosphate oxygens in some of the following figures. However, these interactions play an integral role in phosphate transfer reactions.

Mechanistically speaking, a phosphate transfer reaction at a phosphorus center can be thought of as much like a S_N2 reaction at a carbon center. Like in an SN2 reaction, the nucleophile in a phosphoryl transfer approaches the electrophilic center opposite the leaving group from the back side, as shown in Figure \(\PageIndex{5}\).

Figure \(\PageIndex{5}\): Phosphate transfer mechanism and energy diagram

As the nucleophile gets closer and the leaving group begins its departure, the bonding geometry at the phosphorus atom changes from tetrahedral to trigonal bipyramidal at the pentavalent (5-bond) transition state (Figure \(\PageIndex{6}\)). As the phosphorus-nucleophile bond gets shorter and the phosphorus-leaving group bond gets longer, the bonding picture around the phosphorus atom returns to its original tetrahedral state.  However, the stereochemical configuration has been 'flipped' or inverted.

In the trigonal bipyramidal transition state, the five substituents are not equivalent: the three non-bridging oxygens are said to be equatorial (forming the base of a trigonal bipyramid). In contrast, the nucleophile and the leaving group are said to be apical (occupying the tips of the two pyramids).

Figure \(\PageIndex{6}\): Transition state of the phosphate transfer reaction

Although stereochemical inversion in phosphoryl transfer reactions is predicted by theory, the fact that phosphoryl groups are achiral made it impossible to observe the phenomenon directly until 1978, when a group of researchers was able to synthesize organic phosphate esters in which stable oxygen isotopes ¹⁷O and ¹⁸O were specifically incorporated (Figure \(\PageIndex{7}\)). This created a chiral phosphate center.

Figure \(\PageIndex{7}\): Inversion of stereochemistry in the phosphate transfer reaction

Subsequent experiments with phosphoryl transfer-catalyzing enzymes confirmed that these reactions proceed with stereochemical inversion. (Nature 1978 275, 564; Ann Rev Biochem 1980 49, 877). (The previous excerpt has been adapted from Chemistry LibreTexts.)

We should note that although the charge-separated resonance form shown above contributes to the structure and, therefore, contributes to the resonance hybrid, the phosphate group is almost always shown with a double-bonded oxygen atom to the phosphorous. Note that we have also depicted the mechanism below with our preferred biochemical arrow pushing, where oxygen attack at phosphorous facilitates the deprotonation of the alcohol (R₁OH) by a general base.

Figure \(\PageIndex{8}\): Common arrow pushing for phosphate transfer

ATP would be an extremely poor electrophile with a formal charge of negative four. Therefore, the phosphoryl transfer from ATP, shown for hexokinase (EC 2.7.1.1) in Figure \(\PageIndex{9}\), requires the binding of a magnesium ion with ATP. The magnesium ion partially neutralizes the negative charge, allowing for nucleophilic attack by the oxygen atom of glucose.

Figure \(\PageIndex{9}\): Hexokinase mechanism with phosphate transfer from ATP

A crystal structure of the enzyme was solved with the substrate, glucose-6-phosphate, bound. The magnesium cofactor and a non-hydrolyzable analog of ATP also occupy the active site. In the substrate analog, ANP, the oxygen of the terminal phosphoanhydride bond of ATP is replaced with a nitrogen atom. It allows us to view probable interactions between the enzyme and the substrate analog of the catalytic complex. In the iCn3D image in Figure \(\PageIndex{10}\), ANP and glucose are bound in the active site. The magnesium ion is shown in green.

Figure \(\PageIndex{10}\): Interactive iCn3D image of the catalytic complex of human glucokinase (a hexokinase isoform, 3FGU). (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?EajeSCpF8GM16HYq5

Serine and other Endoproteases

The mechanism for chymotrypsin, a prototypical serine protease, will be presented to explore the different types of acceptable arrow pushing one can show for this nucleophilic acyl substitution mechanism. Only the relevant peptide bond will be shown in the mechanism. For arrow pushing, it is only necessary to focus on this small portion of the molecule shown in Figure \(\PageIndex{15}\).

Figure \(\PageIndex{15}\): Abbreviated chymotrypsin peptide cleavage reaction

The active site of chymotrypsin contains a catalytic triad, three amino acids working together to carry out the reaction that cleaves the peptide bond. The amino acids involved are the aspartate, histidine, and serine residues mentioned earlier (Figure \(\PageIndex{16}\)).

Figure \(\PageIndex{16}\): Serine protease catalytic triad

The deprotonated aspartate side chain increases histidine’s basicity, allowing it to accept a proton from serine in the catalytic mechanism. In some mechanisms, Asp is shown accepting a proton from histidine; however, simplified arrow pushing can be shown without Asp acting as a proton acceptor, which is how the mechanism will be represented here.

In the first stage of the mechanism, histidine deprotonates serine, which acts as a nucleophile and attacks the partially electropositive carbon atom of the carbonyl functional group (Figure \(\PageIndex{17}\)). In the simplest form of arrow pushing shown, histidine deprotonates the nucleophilic using the lone pair on nitrogen, and the electrons from the hydrogen-oxygen bond are shown attacking. The carbonyl double bond breaks, shifting the electrons onto oxygen.

This forms a tetrahedral sp³ hybridized carbon atom from the sp² hybridized carbonyl group, which is why it is called a tetrahedral intermediate.

Figure \(\PageIndex{17}\): First stage of the chymotrypsin mechanism

Using the simple arrow-pushing model again, the electrons from the negatively charged tetrahedral intermediate reform a double bond, kicking out the amine leaving group, which accepts an H+ from protonated histidine, neutralizing the charge on histidine. The arrow from the N-H bond neutralizes the positive charge on histidine; please note that this arrow is essential to show. In all deprotonations, it is conventionally to show electrons from a breaking bond becoming a lone pair on the atom receiving them.

Another acceptable form of arrow pushing for this stage of the reaction requires more arrows but better depicts how the enzyme is interacting with the substrate in the active site (Figure \(\PageIndex{18}\). Because an active site often uses entropy reduction, bringing substrates close together in a reactive orientation, the lone pairs on heteroatoms already interact favorably to form the new bond. Additionally, in the first mechanism, it almost appears that a serine alkoxide attacks the carbon of the peptide bond and that R-NH^- (a poor leaving group) departs the molecule, picking up a proton after the bond breaks.

Therefore, these subtleties are considered in this second mechanism option. In the first step, the proton on serine is deprotonated by histidine, and those electrons are pushed toward the serine oxygen atom. The serine oxygen is positioned close to the carbonyl carbon of the amide bond, and an arrow originating from the serine lone pair depicts the attack. This type of arrow pushing implies that the attack and deprotonation steps are in concert.

Figure \(\PageIndex{18}\): Alternate arrow pushing for the first stage of the chymotrypsin mechanism

In the second step, the electrons that push down from the negatively charged oxygen atom break the N-H bond, becoming a lone pair on oxygen. Simultaneously, the lone pair already present on nitrogen is shown deprotonating histidine.

It is important to note that because biochemical mechanisms are often lengthy, they may be shown with the tetrahedral intermediate omitted. This arrow pushing for the first step, which may be shown using either convention described above, shows the serine oxygen attacking and the amine leaving group departure in one step, as shown in Figure \(\PageIndex{19}\) below. Note that this abbreviated style of arrow pushing, which does not show the tetrahedral intermediate, often uses generic acids and bases, so it does not keep track of protonation states or account for the amino acid residues performing protonation and deprotonation steps (which is quite essential in the chymotrypsin mechanism).

Figure \(\PageIndex{19}\): Abbreviated arrow pushing for the first stage of the chymotrypsin mechanism

The covalent intermediate must be released from the enzyme for chymotrypsin to catalyze another reaction. This second nucleophilic acyl substitution also proceeds through a tetrahedral intermediate; this time, water is the nucleophile, as shown in Figure \(\PageIndex{20}\).

Figure \(\PageIndex{20}\): Second stage of the chymotrypsin mechanism

Now that we've seen the steps in detail let's put all this together to show the full mechanism for serine protease cleavage of protein, shown in Figure \(\PageIndex{21}\).

Figure \(\PageIndex{21}\): General mechanism of peptide bond cleavage by chymotrypsin and other serine proteases

Here a summary of what the Figure \(\PageIndex{21}\) mechanism shows:

• The deprotonated His 57 acts as a general base to abstract a proton from Ser 195, enhancing its nucleophilicity as it attacks the electrophilic C of the amide or ester link, creating the oxyanion tetrahedral intermediate. Asp 102 acts electrostatically to stabilize the positive charge on the His.
• The oxyanion collapses to form a double bond between the O and the original carbonyl C, with the amine product as the leaving group. The protonated His 57 acts as a general acid, donating a proton to the amine leaving group and regenerating the unprotonated His 57.
• The mechanism repeats itself, with water as the nucleophile, which attacks the acyl-enzyme intermediate to form the tetrahedral intermediate.
• The intermediate collapses again, releasing the E-SerO—as the leaving group, which is deprotonated by His 57. This regenerates both His 57 and Ser 195 in the normal protonation state, and the enzyme is now ready for another catalytic round of activity.
• The mechanism for the first nucleophilic attack (by Ser) is the same as for the second (by water). The reverse mechanism of condensation of two peptides would be the reverse of the above mechanism and is an example of the principle of microscopic reversibility.

In short, many of the previously encountered catalytic mechanisms are deployed in chymotrypsin catalysis. These include nucleophilic catalysis (with the Ser 195 forming a covalent intermediate with the substrates), general acid/base catalysis with His 57, and loosely, electrostatic catalysis with Asp 102 stabilizing not the transition state or intermediate, but the protonated form of His 57. An important point to note is that His, as a general acid and base catalyst, stabilizes developing charges in the transition state and provides a path for proton transfer, without which reactions would have difficulty proceeding.

One final mechanism is at work. The enzyme does indeed bind the transition state more tightly than the substrate. Crystal structures with poor "pseudo"-substrates that get trapped as partial tetrahedrally-distorted substrates of the enzyme and with inhibitors show that the oxyanion intermediate, and hence presumably the TS, can form H-bonds with the amide H (from the main chain) of Gly 193 and Ser 195. These cannot be made to the trigonal, sp² hybridized substrate. In the enzyme alone, the hole into which the oxyanion intermediate and TS would be placed is not occupied. This oxyanion hole is occupied in the tetrahedral intermediate.

A crystal structure of a relative of chymotrypsin, trypsin, which cleaves after positively charged lysine and arginine side chains, has been determined with a bound transition state analog inhibitor. The transition state inhibitor is t-butoxy-Ala-Val-boro-Lys methyl ester, as shown in Figure \(\PageIndex{22}\).

Figure \(\PageIndex{22}\): The transition state inhibitor of trypsin, t-butoxy-Ala-Val-boro-Lys methyl ester

Recall from introductory chemistry that neutral boron compounds like BH₃ and BF₃ are trigonal planar (sp²) and electron deficient. Although the boron is not charged, it has a significant partial positive charge (δ⁺) so it is electrophilic. The nucleophilic oxygen of Ser 195 can then attack the boron to form a tetrahedral intermediate. This intermediate is not an oxyanion; one of the attached oxygens with a δ^- charge occupies the oxyanion hole.

Figure \(\PageIndex{23}\) shows the active site group in trypsin interacting with a part of the transition state analog (1BTZ). The serine 195 side chain O is covalently attached to the boron, so the boron is now tetrahedral (sp³).

Figure \(\PageIndex{23}\): Stabilization of a transition state analog by the oxyanion hole

The yellow dotted lines show hydrogen bonding between the backbone amide hydrogens of Ser 195 and Gly 193 with the methoxy oxygen of the now tetrahedral borate transition state analog inhibitor. The boron atom is the yellow/orange sp³ atom connected to 3 oxygen (red) atoms and one carbon (cyan) atom. Normally, the oxyanion O^- from the tetrahedral intermediate in amide or ester cleavage would occupy the oxyanion hole.

Figure \(\PageIndex{24}\) shows an interactive iCn3D model of the active site of the phenylethane boronic acid (PBA) complex of alpha-chymotrypsin (6cha).

Figure \(\PageIndex{24}\): Active site of the phenylethane boronic acid (PBA) complex of alpha-chymotrypsin (6cha). (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...qXUxbrakurYmx6

 

Many enzymes have active site serines which act as nucleophilic catalysts in nucleophilic substitution reactions (usually hydrolysis). One such enzyme is acetylcholinesterase, which cleaves the neurotransmitter acetylcholine in the synapse of the neuromuscular junction (Figure \(\PageIndex{25}\)).

Figure \(\PageIndex{25}\): Reaction catalyzed by acetylcholinesterase

The neurotransmitter leads to muscle contraction when it binds its receptor on the muscle cell surface. The transmitter must not reside too long in the synapse; otherwise, muscle contraction will continue uncontrolled. To prevent this, a hydrolytic enzyme, acetylcholinesterase, a serine esterase found in the synapse, cleaves the transmitter at rates close to diffusion-controlled. Diisopropylphosphofluoridate (DIPF) also inhibits this enzyme, effectively making it a potent chemical warfare agent. Another fluoride-based inhibitor of this enzyme, sarin (Figure \(\PageIndex{26}\)), is the most potent lethal chemical agent of this class known. Only 1 mg is necessary to kill a human being.

Figure \(\PageIndex{26}\): Sarin

Serine Protease Specificity

Serine proteases have unique specificities that allow the cleavage of target proteins after a different subset of side chains. They cleave the peptide bond on the carboxylic acid side of specific amino acids, and the specificity is determined by the size/shape/charge of the amino acid side chain that fits into the enzyme’s S1 binding pocket (Figure \(\PageIndex{27}\)). The pancreatic digestive enzymes, trypsin, chymotrypsin, and elastase are three chymotrypsin-like family members with high sequence homology. The protein cleavage sites of these enzymes vary. Trypsin cleaves proteins on the carboxylic side of basic residues, such as lysine and arginine, while chymotrypsin cleaves after aromatic hydrophobic amino acids, such as phenylalanine, tyrosine, and tryptophan. Elastase cleaves after small, hydrophobic residues like glycine, alanine, and valine. As shown in Figure \(\PageIndex{27}\), variations in the amino acid residues within the binding pocket of these proteases enable electrostatic interactions with the substrate and determine sequence specificity.

Figure \(\PageIndex{27}\): Substrate Specificity of Trypsin, Chymotrypsin, and Elastase. The upper panel shows the space-filling crystal structures of Trypsin, Chymotrypsin, and Elastase, respectively, with the S1 substrate binding pocket indicated. The lower panel depicts the S1 binding domains of each protease in more detail, with the indicated important amino acid R-groups. For Trypsin, an aspartate residue in the lower portion of the S1 pocket aids in electrostatic interactions with basic residues of the substrate. The Chymotrypsin S1 binding pocket is large and hydrophobic, accommodating the substrate's aromatic residues. In contrast, the Elastase S1 binding pocket is small and hydrophobic, only allowing other small and hydrophobic R-groups to dock in this location. Image modified from: Goodsell, D. (2012) Molecule of the Month, Protein Database and Aleia Kim

A schematic nomenclature developed by Berger and Schechter is often used to show the sites on the substrate (labeled P3, P2, P1, P1', P2' and P3') referring to the products made after cleavage of the peptide/protein that is cleaved between P1 and P' (the scissile bond) and the corresponding sites on the protease (S3, S2, S1, S1', S2' and S3'). This is illustrated in Figure \(\PageIndex{28}\).

Figure \(\PageIndex{28}\): Nomenclature of denote key interacting groups in the enzyme (S_n) and in the substrate (P_n) around the scissile bond for protein cleavage

Serine proteases are just one type of endoprotease. However, they are highly abundant in both prokaryotes and eukaryotes. Protease A, a chymotrypsin-like protease from Streptomyces griseus, has a very different primary sequence than chymotrypsin, but its overall tertiary structure is similar to chymotrypsin. The positions of the catalytic triad amino acids in the primary sequences of the protein are very similar, indicating that the genes for the proteins diverged from a common precursor gene. In contrast, subtilisin, a serine protease from B. Subtilis, has both limited sequence and tertiary structure homology to chymotrypsin. However, when folded, it also has a catalytic triad (Ser 221 - His 64 - Asp 32) similar to chymotrypsin (Ser 195 - His 57 - Asp 102). The alignment of the core structures of chymotrypsin (5cha, magenta) and subtilisin (1sbc, cyan) are shown in Figure \(\PageIndex{29}\).

Figure \(\PageIndex{29}\): Convergent Evolution of the Serine Proteases, Chymotrypsin and Subtilisin.

The list of serine proteases is quite long. They are grouped into two broad categories - 1) those that are chymotrypsin-like and 2) those that are subtilisin-like. Though subtilisin-type and chymotrypsin-like enzymes use the exact mechanism of action, including the catalytic triad, the enzymes are otherwise unrelated to each other by sequence and appear to have evolved independently. They are, thus, an example of convergent evolution - a process where the evolution of different forms converges on a structure to provide a common function.

Proteases have multiple functions besides digestion, including degrading old or misfolded proteins and activating precursor proteins (such as clotting proteases and proteases involved in programmed cell death). Four different protease classes have been found based on residues in their active sites. Proteases can also be integral membrane proteins and carry out their activities in the membrane's hydrophobic environment. For example, aberrant cleavage of the amyloid precursor protein by the membrane protease presenilin can lead to the development of Alzheimer's Disease.

Table \(\PageIndex{4}\) below shows a classification of proteases based on their active site nucleophiles.

Class (active site)	Active Site Nucleophile	Location	Examples
Serine/Threonine Hydrolases	Ser/Thr	soluble	trypsin, chymotrypsin, subtilisin, elastase, clotting enzymes, proteasome
		membrane	Rhomboid family
Aspartic Hydrolases	H2O activated by 2 Asps	soluble	pepsin, cathepsin, renin, HIV protease
		membrane	β-secretase (BACE), presenilin I, signal peptide peptidase
Cysteinyl Hydrolases	Cys	soluble	bromelain, papain, cathepsins, caspases
		membrane	?
Metallo Hydrolases	H2O activated by 1 or 2 metal ions	soluble	thermolysin, angiotensin-converting enzyme
		membrane	S2P family

Glutamate Hydrolases	Glu	.	endolysins (fungal)
Asparagine Lysases (EC4) (elimination rx which are self-cleavage and hence not catalytic)	Asn	.	Tsh autotransporter E. Coli

Table \(\PageIndex{4}\): Protease classification

How do integral membrane proteases catalyze the hydrolysis (using water) of transmembrane domains in proteins, given the hydrophobic environment of the bilayer? The rhomboid class of membrane proteases, found in prokaryotic and eukaryotic cells, is one of the most conserved membrane proteins. Instead of using a catalytic triad, these serine proteases use a dyad of Ser 201 as a nucleophile and His 254 as a general acid/base.

The chief requirement for rhomboids' protein substrates is a transmembrane domain in the target protein. No specific amino acid sequence seems required for the specificity of one particular substrate, the Drosophila transmembrane protein Spitz, found in Golgi membranes. When this protein is cleaved, the remaining part is released as a water-soluble protein to the lumen of the Golgi, where it can eventually be released from the cell. The soluble protein fragment released from the cell contains an epidermal growth factor domain.

The structure of a rhomboid protease, GlpG (EC:3.4.21.105), from E. Coli, was determined. It is a serine protease with a catalytic dyad (Ser 201 and His 254) instead of a triad, as in most serine proteases. This transmembrane protein has six transmembrane helices. The enzyme has a polar active site at the bottom of a V-shape opening situated laterally in the membrane. The active site His and Ser residues are deep in this V-shaped cleft, well below the surface of the membrane. Access to the transmembrane strand of the protein substrate is blocked by a loop, which must be gated open to allow substrate access between the V-shaped gap between helices S1 and S3. Ser 201 (nucleophile) and His 254 (general base/acid) are essential for activity. The active site His 254 can be covalently modified with different chloromethylketone peptide derivatives. Figure \(\PageIndex{30}\) shows an interactive iCn3D model of the Rhomboid intramembrane protease GlpG 4QO2.

Figure \(\PageIndex{30}\): Rhomboid intramembrane protease GlpG (4QO2). (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...ft9vFn7WqcujM7

Proteolytic enzymes (peptidases, proteases, and proteinases) are found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role in biological processes and the life cycle of many pathogens. Proteases are extensively applied enzymes in several sectors of industry and biotechnology. Furthermore, numerous research applications require their use, including the production of Klenow fragments, peptide synthesis, digestion of unwanted proteins during nucleic acid purification, cell culturing, and tissue dissociation, preparation of recombinant antibody fragments for research, diagnostics, and therapy, and the exploration of the structure-function relationships.

Proteolytic enzymes belong to the hydrolase class of enzymes and are grouped into the subclass of peptide hydrolases or peptidases. Depending on the site of enzyme action, the proteases can also be subdivided into exopeptidases (like chymotrypsin) or endopeptidases (like carboxypeptidase A), as we will discuss next. Exopeptidases, such as aminopeptidases and carboxypeptidases, catalyze the hydrolysis of the peptide bonds near the substrate's N- or C-terminal ends, respectively. Endopeptidases cleave peptide bonds at internal locations within the peptide sequence. These differences are illustrated in Figure \(\PageIndex{31}\). Proteases may also be nonspecific and cleave all peptide bonds equally, or they may be highly sequence-specific and only cleave peptides after certain residues or within specific localized sequences.

Figure \(\PageIndex{31}\): Peptide bond cleavage by various proteases (Figure from Mótyán, J.A., et al. (2013) Biomolecules 3(4), 923-942).

The action of proteolytic enzymes is essential in many physiological processes. For example, proteases function in the digestion of food proteins, protein turnover, cell division, the blood-clotting cascade, signal transduction, processing of polypeptide hormones, apoptosis, and the life-cycle of several disease-causing organisms, including the replication of retroviruses such as the human immunodeficiency virus (HIV). Due to their key role in the life cycle of many hosts and pathogens, they have significant medical, pharmaceutical, and academic importance.

About 2% of human genes encode proteolytic enzymes necessary in many biological processes. Because proteases have become important therapeutic targets, they are intensively studied to explore their structure-function relationships, investigate their interactions with substrates and inhibitors, develop therapeutic agents for antiviral therapies, improve their thermostability and efficiency, and change their specificity by protein engineering for industrial or therapeutic purposes.

Carboxypeptidase A

This enzyme (EC 3.4.17.1) cleaves the C-terminal amino acid from a protein through a hydrolysis reaction. It is an exoprotease (not an endoprotease, which cleaves proteins internally within the sequence). Regarding selectivity toward C-terminal amino acids, its activity is increased if the C-terminal side chain group is aromatic or branched aliphatic (Phe, Tyr, Trp, Leu, or Ile). X-ray structures of the enzyme with and without a competitive inhibitor show a sizeable conformational change at the active site when an inhibitor or substrate is bound. Without inhibitors, several waters occupy the active site. When an inhibitor (and presumably, by extension, a substrate) is bound, the water leaves (which is entropically favored), and Tyr 248 swings around from near the surface of the protein into the active site to interact with the carboxyl group of the bound molecule, a distance of motion equal to about 1/4 the diameter of the protein. This effectively closes off the active site and expels the water.

A Zn²⁺ ion is present at the active site. It is bound by His 69, His 196, Glu 72, and finally, a water molecule as the fourth ligand. A hydrophobic pocket interacting with the substrate's phenolic group accounts for the protein's specificity. In the catalytic mechanism, Zn²⁺ might have several roles. In one, it may help coordinate water and make it more nucleophilic by either polarizing the water or converting it to a more potent nucleophile OH^-. It might also stabilize developing negative charges in the transition state and in an intermediate. Two possible mechanisms have been offered.

The Water Pathway: In this proposed mechanism, water acts as a nucleophile and is deprotonated by Glu 270, acting as a general base. Glu 270, along with Zn₂₊, helps to promote the dissociation of a proton from the bound water, making it a better nucleophile. Water attacks the electrophilic carbon of the sessile bond, forming a tetrahedral intermediate. The tetrahedral intermediate then collapses, expelling the alkoxy leaving group, which picks up a proton from Glu 270, now acting as a general acid catalyst. People used to believe that Tyr 248 acted as a general acid, but mutagenesis showed that Tyr 248 could be replaced with Phe 248 without significantly affecting the reaction rate. Figure \(\PageIndex{33}\) shows a simplified reaction scheme.

Figure \(\PageIndex{33}\): Water pathway mechanism for carboxypeptide A. After Wu et al. J Phys Chem B. 2010 July 22; 114(28): 9259–9267. doi:10.1021/jp101448j

Nucleophilic Pathway: In this pathway, Glu 270 is the primary initial nucleophile that forms the initial tetrahedral intermediate. The role of Zn²⁺ is in charge stabilization. This pathway is illustrated in Figure \(\PageIndex{34}\).

Figure \(\PageIndex{34}\): Nucleophilic Pathway after Wu et al. J Phys Chem B. 2010 July 22; 114(28): 9259–9267. doi:10.1021/jp101448j

Figure \(\PageIndex{35}\) shows an interactive iCn3D model of the active site of bovine carboxypeptidase in the absence of a substrate or inhibitor (1M4L). The Zn²⁺ ion is shown as a red sphere.

Figure \(\PageIndex{35}\): Bovine carboxypeptidase A (1M4L) (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...sX1NYbGav1CFY6

Note how far Tyr248 is away from the active site in the model. Glu72 and Glu270 are negatively charged in the resting state of the enzyme at pH 7.5. The values are much higher (weaker acid) than the solution pK_a of the side chain of glutamic acid. Also, the water bound to the Zn²⁺ is long enough to suggest that the water is neutral and not in the form of OH^- in this form of the enzyme. If OH^- were present, the distance between it and the Zn²⁺ would be shorter due to the great electrostatic force.

Figure \(\PageIndex{36}\) shows an interactive iCn3D model of the active site of bovine carboxypeptidase bound to the inhibitor aminocarbonylphenylalanine (1HDU). The Zn²⁺ ion is shown as a red sphere.

Figure \(\PageIndex{36}\): Bovine carboxypeptidase A bound to the inhibitor aminocarbonylphenylalanine (1HDU). (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...vWt2qeZtUrwj78

Note the closer proximity of tyrosine 248 to the active site.

Lysozyme

Lysozyme (EC 3.2.1.17), found in cells and secretions of vertebrates but also in viruses that infect bacteria, cleaves peptidoglycan GlcNAc (β-1,4) MurNAc repeat linkages (NAG-NAM) in the cell walls of bacteria and the GlcNAc(β-1,4) GlcNAc (poly-NAG) in chitin, found in the cells walls of certain fungi. Since these polymers are hydrophilic, the enzyme's active site would be expected to contain a solvent-accessible channel into which the polymer could bind. The crystal structures of lysozyme and complexes of lysozyme and NAG have been solved to high resolution. The inhibitors and substrates form strong H bonds and some hydrophobic interactions with the enzyme cleft. Kinetic studies using (NAG)_n polymers show a sharp increase in k_cat as n increases from 4 to 5. The k_cat for (NAG)₆ and (NAG-NAM)₃ are similar. Model studies have shown that for catalysis to occur (NAG-NAM)₃ binds to the active site with each sugar in the chair conformation, except the fourth, which is distorted to a half chair form. This labilizes the glycosidic link between the 4^th and 5^th sugars. Additional studies show that if the sugars that fit into the binding site are labeled A-F, then because of the bulky lactyl substituent on the NAM, residues C and E cannot be NAM, which suggests that B, D, and F must be NAM residues. Cleavage occurs between residues D and E.

A review of the chemistry of glycosidic bond (an acetal) formation and cleavage shows that acids catalyze the acetal cleavage and proceed through an oxonium ion, which exists in resonance form as a carbocation. A reaction mechanism of hemiacetal/acetal formation and cleavage is illustrated in Figure \(\PageIndex{37}\).

Figure \(\PageIndex{37}\): A reaction mechanism of hemiacetal/acetal formation and cleavage

Catalysis by the enzyme involves Glu 35 and Asp 52, which are in the active site. Polar groups surround Asp 52, but Glu 35 is in a hydrophobic environment. This should increase the apparent pK_a of Glu 35, making it less likely to donate a proton and acquire a negative charge at low pH values, making it a better general acid at higher pH values. Here is a possible general mechanism:

• binding of a hexasaccharide unit of the peptidoglycan with concomitant distortion of the NAM.
• protonation of the sessile acetal O by the general acid Glu 35 (with the elevated pK_a) facilitates cleavage of the glycosidic link and the resonance stabilized oxonium ion formation.
• Asp 52 stabilizes the positive oxonium through electrostatic catalysis. The distorted half-chair form of the NAM stabilizes the oxonium, which requires co-planarity of the substituents attached to the sp² hybridized carbon of the carbocation resonant form (much like we saw with the planar peptide bond).
• water attacks the stabilized carbocation, forming the hemiacetal by releasing the extra proton from water to the deprotonated Glu 35, reforming the general acid catalysis.

Part of a mechanism illustrating the roles of Glu 35 and Asp 52 is shown below in Figure \(\PageIndex{38}\).

Figure \(\PageIndex{38}\): Mechanism of acetal cleavage by lysozyme

Binding and distortion of the D substituent of the substrate (to the half chair form as shown above) occurs before catalysis. Since this distortion helps stabilize the oxonium ion intermediate, it presumably stabilizes the transition state. Hence, this enzyme appears to bind the transition state more tightly than the free, undistorted substrate, yet another catalysis method.

pH studies show that side chains with pKa's of 3.5 and 6.3 are required for activity. These presumably correspond to Asp 52 and Glu 35, respectively. If the carboxy groups of lysozyme are chemically modified in the presence of a competitive inhibitor of the enzyme, the only protected carboxy groups are Asp 52 and Glu 35.

In an alternative mechanism, Asp 52 acts as a nucleophilic catalyst and forms a covalent bond with NAM, expelling a NAG leaving group with Glu 35 acting as a general acid as shown in Figure \(\PageIndex{39}\). This alternative mechanism is also consistent with other β-glycosidic bond cleavage enzymes. Substrate distortion is also important in this alternative mechanism.

Figure \(\PageIndex{39}\): Alternative mechanism for lysozyme catalysis employing Asp 52 as a nucleophilic catalyst (after Vocadlo et al., Nature, 412 (2001), https://www.nature.com/articles/35090602.

Recent structural work shows that Asp 52 is involved in a strong hydrogen bond network that might preclude its ability to form a covalent bond with the glycan substrate. An earlier structure (1H6M) did show a covalent bond.

Figure \(\PageIndex{40}\) shows an interactive iCn3D model of the active site of hen egg white lysozyme bound to a (NAG)₄ glycan (7BR5). Note the positions of E35 and D52.

Figure \(\PageIndex{40}\): Interactions of hen egg white lysozyme with bound (NAG)₄ glycan (7BR5) (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?mnZuP4W4pfTxKRmX7

Summary

The chapter introduces the essential concept of arrow pushing as a tool to depict enzyme reaction mechanisms, emphasizing its importance in visualizing the flow of electrons during catalysis. It explains that arrow pushing is not merely a drawing exercise but a method to understand how specific chemical transformations occur within enzyme active sites. The text outlines the conventions of curved arrow notation, detailing how arrows indicate the movement of electron pairs from nucleophiles to electrophiles and illustrate the formation and breaking of chemical bonds.

Key concepts include:

• Electron Flow and Mechanistic Steps: The chapter discusses how the movement of electrons is tracked through each step of an enzymatic reaction, from substrate binding to the formation of intermediates and eventual product release. This approach helps in rationalizing the role of catalytic residues and cofactors in lowering activation energies.

• Interpretation of Reaction Mechanisms: By breaking down complex reactions into individual, understandable steps, the chapter guides students in dissecting enzyme mechanisms. It shows how different reaction intermediates and transition states can be identified and how their stability is crucial for efficient catalysis.

• Application in Biochemical Problem-Solving: The chapter emphasizes that mastering arrow pushing enhances one’s ability to predict reaction outcomes, propose alternative pathways, and understand the impact of mutations or inhibitors on enzyme activity. This skill is fundamental for advanced studies in biochemistry and for research involving enzyme kinetics and molecular enzymology.

Overall, the chapter equips junior and senior biochemistry majors with a robust framework for analyzing and depicting enzyme-catalyzed reactions, fostering a deeper understanding of the molecular underpinnings of catalysis through a detailed and systematic approach to arrow pushing.