Aminotransferases (also called transaminases) are a class of pyridoxal-phosphate-depending enzymes that are known to be at the core of amino acid metabolism in all organisms. Because of their central metabolic position, they have been demonstrated to be involved in a number of diseases that are characterized by inborn errors of metabolism. The best studied of all of this class of enzymes is the aspartate aminotransferase, which equilibrates aspartic acid and glutamic acid through the corresponding a-keto-acids, ketoglutarate and oxalaoceate. The enzyme is a dimer of two identical subunits, each of overall molecular weight about 40,000. The crystal structure of E.coli enzyme shows that each subunit consists of two domains, and that the active site lies at the domain and subunit interfaces. There is a large conformational change, involving the movement of over 100 amino acids in the smaller of the two domains, when substrate binds. Another less well-characterized transaminase is the bacterial D-amino acid transaminase, which does not handle L-amino acids at all but does have s wide specificity for the D-counterparts. Both are crystalline, and both bind substrates in the crystalline state. A third transaminase which will be studied is the alanine specific one from beef heart. The goal of this project is to understand the specificity and catalytic mechanism(s) of these transaminases in terms of their structures. Structural comparison of the aspartate and alanine specific transaminases will give insight into the side-chain specificities of these enzymes. Comparison of the L-and D-specific transaminases will give insight into the chiral specificities of these enzymes. Mutants of the bacterial enzymes and structures of complexes with substrates and inhibitors will giveinsight as to the roles of specific amino acids at the active sites and the structural basis for changes in catalysis (mechanism). The causes of the substrate-dependent conformational change in the aspartate enzyme (and possibly in others) will be probed in detail through the combined use of crystallography and site-specific mutagenesis.
