Pyridoxal phosphate enzymes coenzyme conformation

The chromophoric pyridoxal phosphate coenzyme provides a useful spectrophotometric probe of catalytic events and of conformational changes that occur at the pyridoxal phosphate site of the P subunit and of the aiPi complex. Tryptophan synthase belongs to a class of pyridoxal phosphate enzymes that catalyze /3-replacement and / -elimination reactions.3 The reactions proceed through a series of pyridoxal phosphate-substrate intermediates (Fig. 7.6) that have characteristic spectral properties. Steady-state and rapid kinetic studies of the P subunit and of the aiPi complex in solution have demonstrated the formation and disappearance of these intermediates.73-90 Fig. 7.7 illustrates the use of rapid-scanning stopped-flow UV-visible spectroscopy to investigate the effects of single amino acid substitutions in the a subunit on the rate of reactions of L-serine at the active site of the P subunit.89 Formation of enzyme-substrate intermediates has also been observed with the 012P2 complex in the crystalline state.91 ... 

FIGURE 14.22 Glutamate aspartate aminotransferase, an enzyme conforming to a double-displacement bisnbstrate mechanism. Glutamate aspartate aminotransferase is a pyridoxal phosphate-dependent enzyme. The pyridoxal serves as the —NH, acceptor from glntamate to form pyridoxamine. Pyridoxamine is then the amino donor to oxaloacetate to form asparate and regenerate the pyridoxal coenzyme form. (The pyridoxamine enzyme is the E form.)... 

It is interesting that enzyme reactions of primary and secondary metabolism show the same stereospecificity even if there is no discernible advantage of one mode of stereochemical reaction over the other. Examples are the pyridoxal phosphate-dependent enzymes (C 5) which all have the same stereochemistry of the coenzyme-substrate complex, though an alternative conformer formed by 180° rotation could operate equally well. Hence all pyridoxal phosphate-dependent enzymes obviously evolved from a common ancestral protein in which an arbitrary choice between the two faces of the cofactor was made. 

The coenzyme, pyridoxal 5 -phosphate (PLP), forms an aldimine linkage with the -amino group of Lys-258 via its aldehyde group. PLP also interacts with Asp-222, Tyr-225 and Asn-194 by forming hydrogen bonds thus it maintains proper orientation at the active site. The enzyme is in the open conformation. 

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