Hydrolysis of L-Histidine Methyl Ester

Summary

The hydrolysis of L-histidine methyl ester (HME) by bovine thrombin preparations has been investigated. Activation of purified bovine prothrombin in 25% sodium citrate solution resulted in the simultaneous development of fibrinogen clotting activity and of TAME and HME esterase activities. Most of the HME esterase activity was identified with fibrinogen clotting activity on sequential chromatography of activated prothrombin on DEAE-cellulose and Amberlite CG-50 resin columns, although some HME esterase activity could be demonstrated in concentrates of the autoprothrombin C fraction. The optimum pH for HME hydrolysis by thrombin was found at 7.6 in phosphate and Tris buffered reactions. Tris buffer and other amines depress HME esterase activity of thrombin, while sodium cholate accelerates the reaction. The Michaelis constant, K_m, was estimated to be 0.134 M at pH 7.6 in phosphate buffer and at 37° C.

^* This investigation was supported by the National Heart Institute, National Institutes of Health, under Grant No. HE-10146-02. Some of this material was presented at the Fifteenth Annual Symposium on Blood, Wayne State University School of Medicine, Detroit, Michigan, January 20th and 21st, 1967.

• References

• 1 Cole E.R, Koppel J.L, Olwin J.H. The multiple specificity of thrombin for synthetic substrates. Nature (Lond.) 1967; 213: 405
• 2 Inagami T, Sturtevant J.M. Nonspecific catalyses by alpha-chymotrypsin and trypsin. J. biol. Chem. 1960; 235: 1019
• 3 Laki K, Gladner J.A. Chemistry and physiology of the fibrinogen-fibrin transition. Physiol. Rev. 1964; 44: 127
• 4 Lorand L, Konishi K. Activation of fibrin stabilizing factor of plasma by thrombin. Arch. Biochem. 1964; 105: 58
• 5 Therriault D.G, Gray J.L, Jensen H. Influence of thrombin on rate of prothrombin conversion. Proc. Soc. exp. Biol. (N. Y.) 1957; 95: 207
• 6 Zucker M.B, Borrelli J. Changes in platelet adenosine triphosphate concentration and phosphate distribution during viscous metamorphosis and clot retraction. In Blood Platelets. Little, Brown and Company: Boston, Mass.; 1961: p. 383
• 7 Landaburu B.H, Seegers W.H. Activation of prothrombin. Amer. J. Physiol. 1958; 193: 169
• 8 Rapaport S.I, Shiffman S, Batch M.J, Ames S.B. The importance of activation of antihemophilic globulin and proaccelerin by traces of thrombin in the generation of intrinsic prothrombinase activity. Blood 1963; 21: 221
• 9 Mammen E.F, Thomas W.B, Seegers W.H. Activation of purified prothrombin to auto-pro thrombin I or autoprothrombin II (platelet cofactor II) or autoprothrombin II-A. Thrombos. Diathes. haemorrh. (Stuttg.) 1960; 5: 218
• 10 Neil G.L, Niemann C, Hein G.E. Structural specificity of A-chymotrypsin polypeptide substrates. Nature (Lond.) 1966; 210: 903
• 11 Kauzmann W. Chemical specificity in biological systems. In: Biophysical Science-A Study Program. John Wiley and Sons, Inc.: New York; 1959: p. 549
• 12 Seegers W.H. The purification of prothrombin. Record Chem. Prog. (Kresge-Hooker Sci. Lib.) 1962; 13: 143
• 13 Seegers W.H, Cole E.B, Harmison C.B, Marciniak E. Purification and some properties of autoprothrombin C. Canad. J. Biochem. Physiol. 1963; 41: 1047
• 14 Seegers W.H, Levine W.G, Shepard R.S. Further studies on the purification of thrombin. Canad. J. Biochem. Physiol. 1958; 36: 603
• 15 Roberts P.S. Measurement of the rate of plasmin action on synthetic substrates. J. biol. Chem. 1958; 232: 285
• 16 Roberts P.S. The esterase activities of human plasmin during purification and subsequent activation by streptokinase or glycerol. J. biol. Chem. 1960; 235: 2262
• 17 Ware A.G, Seegers W.H. Two-stage procedure for the quantitative determination of prothrombin concentration. Amer. J. clin. Path. 1949; 19: 471
• 18 Seegers W.H, Smith H.P. Factors which influence the activity of purified thrombin. Amer. J. Physiol. 1942; 137: 348
• 19 Folin O, Ciocalteu V. On tyrosine and tryptophane determinations in proteins. J. biol. Chem. 1927; 73: 627
• 20 Cole E.R, Koppel J.L, Olwin J.H. The hydrolysis of phenylalanine methyl ester by bovine thrombin. Canad. J. Biochem. 1966; 44: 1051
• 21 Curragh E.F, Elmore D.T. Kinetics and mechanism of catalysis by proteolytic enzymes. 2. Kinetic studies of thrombin-catalysed reactions and their modification by bile salts and other detergents. Biochem. J. 1964; 93: 163
• 22 Kerekes M.F. Inhibitory action of glucides on trypsin activity. Nature (Lond.) 1966; 210: 633
• 23 Lineweaver H, Burk D. The determination of enzyme dissociation constants. J. Amer. chem. Soc. 1934; 56: 658
• 24 Sherry S, Troll W. The action of thrombin on synthetic substrates. J. biol. Chem. 1954; 208: 95
• 25 Walsh K.A, Neurath H. Trypsinogen and chymotrypsinogen as homologous proteins. Proc. Nat. Acad. Sci. 1964; 52: 884
• 26 Bender M.L, Kézdy J. Mechanism of action of proteolytic enzymes. Ann. Rev. Biochem. 1965; 34: 49
• 27 Cole E.R. Hydrolysis of L-histidine methyl ester. II. Activity of various proteolytic enzymes with special reference to activators of human plasminogen. Thrombos. Diathes. haemorrh. (Stuttg.) 1968; 19: 334
• 28 Seegers W.H, Cole E.R, Aoki N. Function of Ac-globulin and lipid in blood clotting. Canad. J. Biochem. Physiol. 1963; 41: 2441