Biochemistry: The Chemical Reactions of Living Cells, Volume 2 - D. Metzler 1980

Coenzymes are specialized natural reagents of a unique type.
Thiamine diphosphate
Elucidating a detailed picture of the mechanism of a PLP-dependent enzyme

Let us examine The sequence of distinct stages that an aminotransferase catalytic cycle must go through in ~10^-3 s. First, the substrate binds to the enzyme to form a Michaelis complex. This is followed by a two-stage transaldimination [Equation (8-28)], which in turn is succeeded by the abstraction of the a-hydrogen to yield the quinonoid form of the substrate-PLP imine. Four additional steps are then required for The formation of the ketimine, its Hydrolysis, and the release of the keto acid product, yielding the PMP form of the enzyme. The reaction pathways for certain other Pyridoxal phosphate-dependent Enzymes are even more intricate.

In the case of aspartate aminotransferase, it is logical to assume that the positive charge of the substrate's amino group helps achieve the proper molecular orientation through electrostatic attraction with the negatively charged —O^- group at position 3 of the coenzyme [Equation (8-28)]. Similarly, a positively charged protein residue may attract the substrate's a-СОО^- group within the ES complex and neutralize it. This, in turn, would lower the pK of the substrate's NH₃ group, facilitating proton abstraction [55]. It is tempting to speculate that this proton might then be transferred (possibly via a protein functional group) to the nitrogen of the internal Schiff base C=N group [55]. Thus, the nucleophilic —NН₂ group would become bound through a process that simultaneously enhances the electrophilic character of the imine carbon atom. This would enable the direct addition of the —NН₂ group to —C=N+H, forming the adduct depicted in Equation (8-28).

As pointed out by Ivanov and Karpeisky [55], each step in the overall sequence presumably alters the electronic or steric Properties of the complex as a whole, thereby facilitating the subsequent step. Although unproven, this concept may represent a fundamental principle common to all of enzymology: an enzyme Functions as an efficient catalyst precisely because each successive transformation creates favorable conditions for the next.

Many stages of catalysis by PLP-dependent enzymes require proton transfer, and each such transfer influences the subsequent reaction step. Certain stages involve Conformational Changes in the substrate, the coenzyme, and the enzyme protein alike. For example, The conversion of the adduct in Equation (8-28) into the substrate-coenzyme Schiff base requires a spatial rearrangement, which may involve rotation around a single bond—as illustrated in Equation (8-28)—or a reorientation of the coenzyme itself [33, 35]. Note that the eliminated ε-amino group [Equation (8-28)] is strongly basic. It is frequently suggested that this basic group participates in the subsequent step by abstracting the a-H and transferring it to the 4'-carbon.

PLP can be removed from aspartate aminotransferase and replaced with various coenzyme analogues. While some of these analogues bind tightly, the resulting complexes exhibit low catalytic activity. For instance, the complex formed with N-methylated PLP fails to undergo turnover altogether upon The addition of glutamate. This observation strongly suggests that the proton at the pyridine nitrogen must be abstracted at a specific point in the reaction cycle as an obligatory component of the catalytic mechanism.

The reader should consider what Steric hindrances might arise during the reaction sequence and evaluate the likelihood of forming an adduct with the S-configuration rather than the R-configuration in reaction (8-28).