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B6db references: 5650370

type Journal Article
authors Ayling JE, Snell EE
title Mechanism of action of pyridoxamine pyruvate transaminase
journal Biochemistry
sel unselected
ui 5650370
year (1968)
volume 7
number 5
pages 1616-25
abstract Crystalline pyridoxamine pyruvate transaminase was employed as a model system for study of the kinetics and mechanism of transamination. This enzyme has no coenzyme requirement and catalyzes the reversible reaction: pyridoxamine (A) + pyruvate (B) pyridoxal (C) + L-alanine (D). Initial rate studies showed that the forward reaction followed the rate equation: v = Fm/(1 + Xa/[A] + *b/[B] + 7W[A][B]). The several mechanisms which conform to this equation were distinguished by the following criteria, (a) The type of product inhibition: pyridoxal inhibited competitively with respect to pyridoxamine, and vice versa; all other product inhibitions were not of the competitive type. This is consistent only with a mechanism (eq i) involving E + A W + B) EXY ks(-O\ W + D) EC E + C (i) ordered addition of substrate to enzyme (E) in which pyridoxamine or pyridoxal combines first, followed by formation of a ternary complex (EXY). (b) The equilibrium constant: "eq determined from the Haldane equation for this mechanism, 1.53, agreed closely with the directly determined value of 1.21. (c) The relationship between kinetic parameters for forward and reverse reaction was consistent with the postulated mechanism, (d) The degree of product in hibition: the velocity constants calculated from experimentally obtained kinetic parameters were used to calculate the per cent inhibition expected on addition of a given concentration of product. Calculated and observed values were in good agreement. Direct spectrophotometric measurement of the rate of binding of pyridoxal to enzyme in a stopped-flow apparatus, and of the dissociation constants of the enzyme-pyridoxal and enzyme-pyridoxamine complexes, gave values that agreed with those calculated from steady-state kinetics. This result indicates that the rate law based on steadystate assumptions is valid and provides an independent proof that pyridoxamine (and pyridoxal in the reverse reaction) is the first substrate to combine with enzyme. It also shows that the enzyme-pyridoxal complex measured in the stopped-flow apparatus is either a true reaction intermediate or is in rapid equilibrium with the true intermediate. The mechanism of the pyridoxamine pyruvate transaminase reaction established by these studies, in which pyridoxal and pyridoxamine are substrates, is analogous to that proposed previously for other transaminase reactions in which pyridoxal phosphate and pyridoxamine phosphate serve as coenzymes with the major difference that the coenzymes of the latter reactions do not leave the enzyme surface following the isomerization and cleavage of the enzyme-bound azomethine intermediates.
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