The mechanism of transamination. Function of the histidyl residue at the active site of supernatant aspartate transaminase.

Controlled photooxidation inactivates supernatant glutamate aspartate transaminase through the destruction of histidyl residues only. Since previous studies have failed to show alterations in the over-all enzyme structure, the mechanism of transamination has been examined before and after photooxidation, taking advantage of the spectral changes associated with the enzyme prosthetic group, pyridoxal phosphate, after substrate or inhibitor binding. By spectrophotometric titration of the active site, photooxidized enzyme retains its ability to bind the dicarboxylic acids, glutarate, oxaloacetate, and a-ketoglutarate, to form noncovalent enzyme-substrate complexes. Binding of (Ymethyl aspartate was also measured spectrophotometrically. Because the interaction of the enzyme with radioactive substrate can be trapped by reduction of the enzyme-substrate complex with sodium borohydride, it is shown that aspartate in the forward reaction and a-ketoglutarate in the back reaction form the initial aldimine and ketimine complexes, respectively. The affinity for each substrate or substrate analogue is greater in the case of photooxidized enzyme under normal buffer conditions. Native enzyme exhibits relatively large changes in the dissociation constant with changes in the buffer anion concentration, while photooxidized enzyme fails to show these competitive anion effects. Photooxidized enzyme forms all of the visible enzyme substrate intermediates characteristic of the halftransamination reaction between an amino acid and its keto acid analogue, except for the semiquinoid intermediate which absorbs at 490 mp. This inability correlates with the failure to catalyze the exchange of the a-hydrogen of the amino acid with tritium from tritiated water. From these data and from studies on the rate of the forward and back reaction with cysteine suEnate and Lu-ketoglutarate, respectively, it is concluded that the primary defect after photooxidation of the active site histidine of supernatant glutamate aspartate transaminase occurs in the removal of the a-hydrogen from the aldimine Schiff’s base in the forward reaction. A role of proton acceptor is proposed for the histidine residue at the active site. The identification of amino acids which comprise the active site of an enzyme and the assignment of specific roles to them are essential for an understanding of the mechansim of enzymatic catalysis. The assignment of roles to amino acid residues at the active site has generally been based on studies of the pH dependence of kinetic parameters or on experiments involving chemical modification of these amino acids. The former method is presumptive, in that assignment of a pK to a specific amino acid in a protein involves considerable suppositions. Although chemical modifications more clearly define an amino acid as essential, much difficulty is encountered in assigning a role, since the product of such modification may be totally inactive enzyme. This difficulty has been circumvented in some cases by the use of minimally altered enzymes, whose activity, although decreased, is not destroyed. This is exemplified by the works on carboxypeptidase (1) and chymotrypsin (2). Another approach is possible when an enzyme is characterized by visible changes upon reaction with substrate. For example, Miller and Schwert (3) have shown that destruction of presumed histidine residues results in the inability of lactic dehydrogenase to form the highly fluorescent enzyme-NADH-oxaloacetate complex. Supernatant glutamate aspartate transaminase (EC 2.6.1 .‘I) should be particularly useful for modification studies. Considerable detail of its mechanism of catalysis is known (4). The overall reaction is separable into half-reactions, as shown in Scheme I, where the over-all reaction between aspartate, a-ketoglutarate, glutamate, and oxaloacetate is represented as the sum of two half-reactions, A and B, each of which may be readily studied individually. The unique spectral properties of pyridoxal phosphate enzymes, and of aspartate transaminase, in particular, allow several of the catalytic steps of each half-reaction to be observed separately and directly. In this manner, pyridoxal phosphate acts as a natural reporter of events occurring at the active site. The detection of these events is independent of the ability of the enzyme to carry out the over-all catalysis. The