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Biology LibreTexts

Multi-Substrate Enzymes

How do you resolve the enzymes kinetics of these more complicated systems? The answer is fairly straightforward. You keep one of the substrates (B, for example) fixed, and vary the other substrate (A) and obtain a series of hyperbolic plots of vo vs A at different fixed B concentrations. This would give a series of linear 1/v vs 1/A double-reciprocal plots (Lineweaver-Burk plots) as well. The pattern of Lineweaver-Burk plots depends on how the reactants and products interact with the enzyme.

Sequential Mechanism

In this mechanism, both substrates must bind to the enzyme before any products are made and released.The substrates might bind to the enzyme in a random fashion (A first then B or vice-versa) or in an ordered fashion (A first followed by B). An abbreviated notationscheme developed by W.W. Cleland is shown below for the sequential random and sequential ordered mechanisms. For both mechanisms, Lineweaver-Burk plots at varying A and different fixed values of B give a series of intersecting lines. Derivative curves can be solved to obtain appropriate kinetic constants.

01 sequential.GIF

Ping-Pong Mechanism

In this mechanism, one substrate bind first to the enzyme followed by product P release. Typically, product P is a fragment of the original substrate A.The rest of the substrate is covalently attached to the enzyme E, which is designated as E'. Now the second reactant, B, binds and reacts with the enzyme to form a covalent adduct with the A as it is covalentattached to the enzyme to form product Q. This is now released and the enzyme is restored to its initial form, E. This represents a ping-pong mechanism. An abbreviated notation scheme is shown below for the ping-pong mechanisms. For this mechanism, Lineweaver-Burk plots at varying A and different fixed values of B give a series of parallel lines. An example of this type of reaction might be low molecular weight protein tyrosine phosphatase against the small substrate p-initrophenylphosphate (A) which binds to the enzyme covalently with the expulsion of the product P, the p-nitrophenol leaving group. Water (B) then comes in and covalently attacks the enzyme, forming an adduct with the covalently bound phosphate releasing it as inorganic phosphate. In this particular example, however, you cannot vary the water concentration and it would be impossible to generate the parallel Lineweaver-Burk plots characteristic of ping-pong kinetics. 


 

02 pingpong.gif