Linkage between proton binding and amidase activity in human gamma-thrombin.
The amidase activity of human gamma-thrombin has been studied in the pH range 6-10 as a function of NaCl concentration and temperature. As recently found for human alpha-thrombin [Di Cera, E., De Cristofaro, R., Albright, D.J., & Fenton, J.W., II (1991) Biochemistry 30, 7913-7924], the Michaelis-Menten constant, Km, shows a bell-shaped dependence over this pH range with a minimum around pH 7.9 in the presence of 0.1 M NaCl at 25 degrees C. The catalytic constant, kcat, has a bell-shaped pH dependence with a maximum around pH 8.6. A thermodynamic analysis of these parameters has enabled a characterization of the linkage between proton and substrate binding, its dependence on NaCl concentration, and the relevant entropic and enthalpic contributions to binding and catalytic events. Three groups seem to be responsible for the control of gamma-thrombin amidase activity as a function of pH. One of these groups has pK values that are significantly different from those found for alpha-thrombin, and all groups show slightly perturbed enthalpies of ionization. The dependence of gamma-thrombin amidase activity on NaCl concentration is different from that of alpha-thrombin. Increasing NaCl concentration always decreases the substrate affinity for the enzyme in the case of alpha-thrombin, regardless of pH. In the case of gamma-thrombin, such an effect is observed only in the pH range 7.5-9, and a reversed linkage is observed at pH less than 7 and greater than 9.5.(ABSTRACT TRUNCATED AT 250 WORDS)
De Cristofaro
R
R
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Fenton
J W
JW
Di Cera
E
E
D000581:Amidohydrolases / Q000737:chemistry*; D001665:Binding Sites / Q000187:drug effects; D002384:Catalysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011485:Protein Binding; D011522:Protons; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D013917:Thrombin / Q000737:chemistry*
D011522:Protons; D012965:Sodium Chloride; D013917:Thrombin; D000581:Amidohydrolases; C019665:amidase