SUBSITE SPECIFICITY STUDIES ON THE UNUSUAL CYSTEINE PROTEASE CLOSTRIPAIN - CHARGED RESIDUES IN THE P-3 POSITION INDICATE A NARROW SUBSITE REGION

The importance of electrostatic interactions between charged residues at the P-3 position of substrates and the S-3 subsite of the cysteine protease clostripain was investigated, For this purpose quantitative e nzymatic hydrolysis studies using steady state kinetics have been carr ied out within a set of N-alpha-protected synthetic dipeptide ester su bstrates with systematic changes of their charge in the P-3 position, It was demonstrated that, in contrast to the former postulated second anionic S-3 subsite, the lowest specificity was for the hydrolysis of the positively charged substrates. However, this effect was strongly d ependent on the individual amino acid at P-1. Furthermore, we investig ated how far these P-3-S-3 interactions reflect on the S' subsite spec ificity via acyl transfers. Apart from the general weak influence of t he charge at P-3 on the deacylation kinetics, nucleophiles with prolin e at P-1' play an extraordinary role. Surprisingly, in contrast to the poor primary lysine specificity, acyl transfer using P-1 lysine subst rates does not affect the nucleophile efficiency found with the corres ponding arginine substrates.