The esterolytic specificity of bovine thrombin and Factor Xa

Steady state kinetics are compared for the hydrolysis of t-butoxycarbonyl-L-lysine methyl ester and several peptidyl lysine methyl esters catalysed by bovine thrombin and Factor Xa. Thrombin-catalysed reactions have lower Km values and higher kcat/Km values than do reactions catalysed by Factor Xa. Values of kcat are comparable and do not show any particular trend. The best substrate in the present series was t-butoxycarbonylglycylglycyl-L-lysine methyl ester. Thrombin and Factor Xa may possess a hydrophobic region near the P2 binding site which is unfavourable for either asparagine or D-alanine but which readily accommodates glycine, L-alanine or L-phenylalanine. The major improvement in Factor Xa hydrolysis occurred with the occupation of the P2 site by an amino residue while for thrombin the major improvement occurred with the occupation of the P3 site.