Ribonuclease structure and catalysis: effects of crystalline enzyme, alcohol cryosolvents, low temperatures, and product inhibition.
In order to determine the necessary conditions to stabilize intermediates in ribonuclease A catalysis at subzero temperatures for structural studies, we have examined the suitability of alcohol-based cryosolvents. On the basis of thermal denaturation transition curves, the enzyme is in the native conformation in high concentrations of ethanol and methanol, provided the temperature is suitably low. The effects of methanol on the catalytic properties for the hydrolysis for mono- and dinucleotide substrates also are consistent with the absence of adverse effects of the cosolvent. Significant methanolysis occurs in the presence of methanol as cosolvent. The kinetics of 2',3'-CMP hydrolysis are complicated by severe competitive product inhibition, both in aqueous and in methanolic solvents, accounting for the previously observed effect of substrate concentration on the observed Km. Computer-aided analysis allowed the determination of the inhibition constant as a function of experimental parameters. The reaction of ribonuclease A with 2',3'-CMP was investigated at subzero temperatures. The turnover reaction could be made negligible at temperatures below -60 degrees C at pH 3-6 in 70% methanol and below -35 degrees C at pH 2.1. The rate of the catalytic reaction with crystalline enzyme was compared to that of enzyme in solution for both 2',3'-CMP and the dinucleotide CpC. The rates were 50- and 200-fold slower, respectively, in the crystal. These investigations allowed calculation of the necessary conditions for NMR and X-ray diffraction experiments on the trapped enzyme--substrate intermediate.
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Division of Natural Sciences, University of California, Santa Cruz 95064.
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D003460:Crystallization; D003568:Cytidine Monophosphate / Q000494:pharmacology; D000431:Ethanol / Q000494:pharmacology; D005615:Freezing; D007700:Kinetics; D000432:Methanol / Q000494:pharmacology; D011489:Protein Denaturation; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D013816:Thermodynamics
D000431:Ethanol; D012259:Ribonuclease, Pancreatic; D003568:Cytidine Monophosphate; D000432:Methanol