A cryo-enzymological study of the catalytic mechanism of carboxypeptidase A using ¹³C NMR

Abstract

Carboxypeptidase A is classified as an exopeptidase enzyme because it is able to remove single hydrophobic amino acid residue from peptide substrates. The proposed mechanisms for the catalytic hydrolysis are still very controversial, although recent findings strongly suggest a noncovalent process. Several cryo-enzyme studies have shown that at lower temperatures, a single intermediate does appear to accumulate to reasonably high levels. Data collected on this intermediate has not been able to establish its identify. In an attempt to obtain unambiguous information on this species, ('13)C NMR was used to study chemical changes taking place at the reactive site on the substrate. Methods were developed to synthesize milligram quantities of specifically labeled substrates and inhibitors from readily available ('13)C-enriched precursors. Cryo-solvent systems were then tested for their ability to (1) lower freezing points (2) dissolve the enzyme (3) maintain the enzyme's native conformation (4) dissolve several turnovers of substrate (5) minimize the solvent viscosity. Three separate cryo-solvent systems were produced which were able to fulfill most of these requirements. An enzyme-substrate experiment using a methanol/ethylene glycol cryo-solvent was performed at -61 (+OR-) 3(DEGREES)C in an NMR spectrometer. The rate of hydrolysis, as determined from the NMR signals, was about two turnovers per day at this temperature. Although adequate signal-to-noise could be obtained, the only signals found during the course of the reaction were product and substrate peaks. No ('13)C signals were found for any metastable product, any enzyme-bound intermediate or any of the enzyme's own natural abundance resonances. Subsequent studies with an enzyme-bound inhibitor species confirmed earlier suspicions that an enzyme-bound nuclei would not give a detectable ('13)C signal under useful cryo-conditions. Further studies showed that the source of the unfavorable relaxation parameters was both the solvent viscosity and protein aggregation. Strategies for dealing with these problems are discussed.