Solvent isotope effects have been examined for the action of the zinc-containing metalloenzyme carboxypeptidase A on ester and peptide substrates. The kinetic parameters for the carboxypeptidase-catalyzed hydrolysis of an ester, O-(trans-cinnamoyl)-L-β-phenyllactate, in 0.05 M Tris-DCl buffer containing 0.5 M NaCl at pD 8.07 and 25° were compared with those obtained from measurements done in 0.05 M Tris-HCl buffer containing 0.5 M NaCl at pH 7.52 and 25°. A (kcat)H2O/(kcat)D2O ratio of approximately 2 was obtained. The value of the Michaelis constant Km was unaffected by the change in solvent as was the inhibition constant, Ki, found for the product, L-β-phenyllactate, which is a competitive inhibitor. These results indicate that a catalytic step involving general base catalysis is probably important in the carboxypeptidase-catalyzed hydrolysis of an ester. A similar set of experiments carried out on the peptide substrate, N-(N-benzoylglycyl)-L-phenylalanine gave ambiguous results. The role of the zinc ion in the catalytic action of carboxypeptidase A can be considered in the light of these findings.
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机译：对于含锌的金属酶羧肽酶A在酯和肽底物上的作用，已经研究了溶剂的同位素效应。将羧肽酶催化的酯O-（反式肉桂酰基）-L-β-苯基乳酸的水解动力学参数在pH值为8.07和25°的0.05 M Tris-DCl缓冲液（含0.5 M NaCl）中进行了动力学参数比较。测量是在0.05 M Tris-HCl缓冲液中进行的，该缓冲液含有0.5 M NaCl，pH为7.52，温度为25°。得到的（kcat） H2O /（kcat） D2O 比约为2。米氏常数Km的值不受溶剂变化的影响，而对于竞争性抑制剂L-β-苯基乳酸的产物的抑制常数Ki也不受其影响。这些结果表明，涉及常规碱催化的催化步骤在羧肽酶催化的酯水解中可能是重要的。在肽底物N-（N-苯甲酰基甘氨酰基）-L-苯丙氨酸上进行的一组类似实验给出了模棱两可的结果。根据这些发现，可以考虑锌离子在羧肽酶A的催化作用中的作用。
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