Roy, R. Sinha and Imperiali, B. (1997) Pyridoxamine-amino acid chimeras in semisynthetic aminotransferase mimics. Protein Engineering, 10 (6). pp. 691-698. ISSN 0269-2139 http://resolver.caltech.edu/CaltechAUTHORS:20120119-093536025
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The transaminase activity of two new semisynthetic RNase-S proteins incorporating a pyridoxamine moiety at the active site has been evaluated. A chemically competent derivative of pyridoxamine phosphate was incorporated into the C-peptide fragments of these non-covalent protein complexes 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 and was introduced instead of Phe8 into the C-peptide sequence via standard solid phase methodology. The two semisynthetic Pam-RNase constructs were designed to probe whether the native ribonuclease catalytic machinery could be enlisted to modulate a pyridoxamine-dependent transamination reaction. Both RNase complexes, H1SP and S1SP, exhibited modest rate enhancements in the Cu(II)-assisted transamination of pyruvate to alanine under single turnover conditions, relative to 5'-deoxypyridoxamine and the uncomplexed C-peptide fragments. Furthermore, multiple turnovers of substrates were achieved in the presence of added L-phenylalanine due to recycling of the pyridoxamine moiety. The modest chiral inductions observed in the catalytic production of alanine and the differences in reactivity between the two proteins could be rationalized 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 structure/ function relationships in a native-like peptidyl architecture.
|Additional Information:||© 1997 Oxford University Press. Received December 1, 1996; revised February 4, 1997; accepted February 11, 1997. This research was supported by the NIH (GM 53098). The authors acknowledge Michael Shogren-Knaak for the synthesis of 59-deoxypyridoxamine.|
|Subject Keywords:||unnatural amino acids; ribonuclease; enzyme mimic; aminotransferase; pyridoxamine; semisynthesis|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Tony Diaz|
|Deposited On:||19 Jan 2012 19:27|
|Last Modified:||19 Jan 2012 19:27|
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