| Popis: |
The knowledge of the pH and ionic strength dependence of kcat and Km for the hydrolysis of S2238 (HD-phe-pip-arg-pNA.2HC1) by alpha-thrombin is essential in determining optimal reaction conditions of residual enzyme in systems where also protease inhibitors and glycosaminoglycan catalysts play a role.We studied the kinetic behavior of S2238 in piperazine/glycylglycine/NaOH buffers from pH 6 to 11 and with a calculated ionic strength up to 0.7 M taking into account the pH-dependent concentration of the buffer species. The kinetic parameters of 60 Michaelis-Menten substrate functions were used for the setup of ionic strength and pH profiles. The kcat values are dependent upon the ionic strength, increasing steeply up to about 0.3 M and decreasing again at high ionic strength.The Km however,reflecting the affinity between enzyme and substrate,is nearly unaffected.The Km values at very alkaline pH are markedly elevated,indicating a conformational form which does not readily bind substrate.The pH profiles for kcat and kcat/Km are displaced towards the low pH side with increasing ionic strength.The ascending limb corresponds to the pK of the Asp-His charge relay system,decreasing with increasing ionic strength from 7.2 to 6.6 in the ES complex and from 6.8 to 6.6 in the free enzyme.Apparently substrate binding provokes a pK increase of the active His residue.The descending limb in the kcat profile could be described by a hypothetical pK varying from 11.5 to 10.7 but the activity decrease is probably due to enzyme inactivation.The alkaline limb of the kcat/Km profile is governed by a pK of 9.4 which is rather independent of the ionic strength and could be attributed to the B-chain terminal isoleucine ,forming a salt bridge with Asp 194 and stabilizing the active site conformation as proven for other serine proteases.Data analysis via a modified Debije function with appropriate estimates for the dielectric constant and the radius of the macro-ion can provide information on the charge density of the enzyme. |
