Pancreatic ribonuclease A is an enzyme that binds up ribonucleic acid (RNA) along the multiple binding subsites that essentially recognize the negatively charged phosphates of the substrate. It is endoribonuclease that catalyse depolimerization of single-stranded RNA. This work gives additional support to the existence of the phosphate-binding site p2 and confirms the central role of Lys-7 in establishing and electrostatic interraction with a phosphate group of the substrate. In this work catalytic properties of recombinant ribonuclease K7H have been studied. This enzyme is a mutant enzyme which contains histidine instead of lysine in a position 7, amino-acid that participates in the main catalytic center of RNase A, named p1. It was obtained by site-directed mutagenesis. Kinetic parameters of K7H have determined with C > p i poli (C) as substrates at pH 5.5 i 7.5. Kinetic parameters of K7H for C > p and as a substrate at pH 5.5 have not altered, but at pH 7.5 were significantly increased. Value Km was also increased, that indicates decreasing of affinity. Increasing of catalysis was double. Results of kinetic parameters of K7H with poli (C) as a substrate in pH 5.5 have shown slight difference according to kinetic parameters of commercial RNase A with poli (C). Significant decreasing of values of all kinetic parameters for K7H were reaction at pH 7.5.

Pancreatic ribonuclease A (RNase A) is a endonuclease that catalyzes depolymerization of ribonucleic acid (RNA) releasing oligonucleotides. In the process of binding enzyme with substrate are involved several non-catalytic phosphate binding subsites, one of them is p2, additional to main catalytic site p1. RNaza A prefers binding and cleavage of longer substrate molecules, and 3',5'-phosphodiester bond should be some six-seven residues apart from the end of molecules of the chain of RNA. In this work is analysed endonuclease activity of recombinant pancreatic RNase A (K7H), that in position seven instead of a lysine there is a histidine, amino acid residue that participates in main catalytic site p1. Mutant enzyme is obtained by site-directed mutagenesis by Kunkel. Results of this investigation have shown that substitution of lysine by histidine in position seven of RNase A has produced total deletion of p2 subsite, and K7H has lost endonuclease activity, and has become exonuclease. These results confirm central role of Lys-7 in establishing p2 subsite and endonuclease activity of pancreatic RNase A.