The Roles of Tyr70 and Tyr225 in Aspartate Aminotransferase Assessed by Analysing the Effects of Mutations on the Multiple Reactions of the Substrate Analogue Serine O‐Sulphate

Abstract

Aspartate aminotransferase catalyses multiple reactions of the glutamate analogue, serine 0-sulphate. The predominant reaction is p-elimination of sulphate to give aminoacrylate (k,,, = 13 s-' for the Escherichia coli enzyme) which may either hydrolyse to pyruvate and ammonia, or react covalently with the enzyme and inactivate it (k,,,,,, = 1.1 XlO-' s-I). Serine 0-sulphate also undergoes a transamination reaction that converts the enzyme to its pyridoxamine form (k,,, = 0.11 s-I). Tyr70 and Tyr225, each of which forms a hydrogen bond with the coenzyme, were substituted with methionine and phenylalanine, respectively. The Y225F mutation does not affect p-elimination but reduces the rates of transamination and inactivation about 70-fold and 3-fold, respectively. Apparently, Tyr225 is not essential for the steps leading to and including abstraction of the proton from Cn of the substrate. It is argued that the Y225F mutation interferes with ketimine hydrolysis. The Y70M mutation affects all three reactions, p-elimination being about fourfold slower, transamination 340-fold slower, and inactivation being 1.4 times faster than in the wild-type enzyme. It is proposed that a hydrogen bond from Tyr70 positions Lys258 for protonation of the quinonoid intermediate at C4' and that, although the full kinetic contribution of this interaction is only revealed in the multiple reactions of serine 0-sulphate, the same interaction is equally important in increasing the reaction specificity for transamination of the natural substrates.Keywords: aspartate aminotransferase ; mutagenesis ; mechanism ; tyrosine ; serine 0-sulfate.Aspartate aminotransferase (AspAT) catalyses the reversible transfer of an amino group from aspartate or glutamate to the 0x0 acids 2-oxoglutarate or oxaloacetate. The enzyme has been purified from many sources and spatial structures have been determined for pig cytosolic [l ] and chicken mitochondria1 AspAT [2-41. Recently, detailed accounts of the structure of AspAT from Escherichia coli [5, 61 have supplemented and amplified earlier descriptions of the enzyme from this source [7, 81. On the basis of extensive work by many authors, involving chemical, spectroscopic, kinetic, and crystallographic studies, a detailed mechanism of enzymic transamination has been proposed [9, 101. The reaction consists of many elementary steps in which both the geometry and the electron distribution of the substrate-coenzyme complex undergo successive changes. A full description of this enzyme requires understanding of the parts played by individual active-site residues in promoting these changes.If serine 0-sulphate, a close analogue of glutamate, is added to AspAT, multiple simultaneous reactions are catalysed [ll]. All these reactions (Scheme 1) are initiated by the abstraction of a proton from the a-carbon atom of the substrate. In the reaction with natural substrates, abstraction of this proton is followed almost exclusively by reprotonation at C4' of the coenzyme leading to transamination. With serine 0-sulphate as sub...