PYRIDOXAMINE AMINO-ACID CHIMERAS IN SEMISYNTHETIC AMINOTRANSFERASE MIMICS

The transaminase activity of two new semisynthetic RNase-S proteins in corporating a pyridoxamine moiety at the active site has been evaluate d, A chemically competent derivative of pyridoxamine phosphate was inc orporated into the C-peptide fragments of these non-covalent protein c omplexes in the form of an unnatural coenzyme-amino acid chimera, 'Pam '. The chimeric Pam residue integrates the-heterocyclic functionality of pyridoxamine phosphate into the side chain of an alpha-amino acid a nd was introduced instead of Phe8 into the C-peptide sequence via stan dard solid phase methodology. The two semisynthetic Pam-RNase construc ts were designed to probe whether the native ribonuclease catalytic ma chinery could be enlisted to modulate a pyridoxamine-dependent transam ination reaction, Both RNase complexes, H1SP and S1SP, exhibited modes t rate enhancements in the Cu(II)-assisted transamination of pyruvate to alanine under single turnover conditions, relative to 5'-deoxypyrid oxamine and the uncomplexed C-peptide fragments. Furthermore, multiple turnovers of substrates were achieved in the presence of added L-phen ylalanine due to recycling of the pyridoxamine moiety, The modest chir al inductions observed in the catalytic production of alanine and the differences in reactivity between the two proteins could be rationaliz ed by the participation of a general base (His12) in complex H1SP, and by the increased tolerance for large amino acid substrates by complex S1SP, which contains serine at this position, The pyridoxamine-amino acid chimera will be useful in the future for examining the coenzyme s tructure/function relationships in a native-like peptidyl architecture .