K. E. T. Kerling

Enzymatic hydrolysis of 2',3'-cyclic CMP by homohistidine-12-ribonuclease S'.

Summary Two semisynthetic RNase S′ analogues, in which the active site residue histidine-12 is replaced by homohistidine, while in one of them methionine-13 is substituted by isoleucine as well, were studied with respect to their capacity to hydrolyze 2′,3′-cyclic CMP, a model substrate for the second step of the RNase catalyzed breakdown of RNA. Kinetic parameters have been determined at pH 6. From the results obtained it is concluded that replacement of histidine-12 by homohistidine has no drastic effect on the catalytic properties of the enzyme in the second step of the reaction.

Synthesis and enzymatic activity of an RNase S′ analogue in which the 4-imidazolylglycyl residue takes the position and the role of histidine-12☆

It is generally recognized that histidine-12 plays a direct role in the mechanism of action of ribonuclease A. Thanks to the S-peptide/S-protein system of Richards [l] it has been possible to study the function of the imidazole-12 moiety in the enzymatic reaction by preparing RNase S’ analogues in which histidine-12 is replaced by other residues. Replacement of L-histidine12 by the isosteric P-pyrazolyl-3-Lalanine [2] or 4-fluoro-Lhistidine [3], both having a p@s of 2.5, results in complete loss of catalytic activity, whereas the binding to Sprotein remains practically unaffected. These findings demonstrate the influence of the pK!Jn on the catalytic efficiency (pKp His 6.0). Recently we reported a remarkably high activity of a RNase S’ analogue in which His-l 2 is replaced by Lhomohistidine [4,5] , that suggestively has almost the same p@ value as histidine. The binding capacity of this S-peptide analogue is lowered by a factor of approximately 100 relative to S-peptide l-20, indicating the influence of lengthening the side chain. We now wish to report an extension of the investiga-