Regulation of carboxypeptidase E. Effect of pH, temperature and Co2+ on kinetic parameters of substrate hydrolysis.
Carboxypeptidase E is a member of the carboxypeptidase A and B gene family, with many of the putative active-site and substrate-binding residues conserved between these enzymes. However, the pH optimum of carboxypeptidase E is substantially lower than that of carboxypeptidases A and B. To evaluate whether the difference in the pH optima of these carboxypeptidases reflects fundamental differences in the ionization behaviour of active-site residues, the influence of pH on carboxypeptidase E activity was examined. The V(max) for hydrolysis of dansyl-Phe-Ala-Arg is pH-independent between 5 and 7, but decreases at pH values below 5. The pKa for the group the protonation of which leads to the loss of activity is approximately 4.8, and the slope of the V(max.)/pH profile suggests that only a single ionizable group is involved. In contrast, Km and V(max.)/Km are dramatically influenced by pH over the range 5-7, with multiple ionizable groups detected in this pH range. The pKa of the group the protonation of which decreases the V(max.) of substrate hydrolysis is lower (4.5) for carboxypeptidase E which had been reconstituted with Co2+. The enthalpy of ionization of the group observed in the V(max.) profile for carboxypeptidase E is approx. 28.9 kJ/mol. These results are compatible with the active-site model of the homologous carboxypeptidase A: in this model the ionization of a metal-bound water molecule is responsible for the observed decrease in V(max.).
Greene
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D
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461.
Das
B
B
Fricker
L D
LD
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000235:genetics* / Q000378:metabolism; D002413:Cations, Divalent; D002417:Cattle; D003035:Cobalt / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature
D002413:Cations, Divalent; D003035:Cobalt; D002268:Carboxypeptidases; D043423:Carboxypeptidase H