[Catalytic properties of recombinant pancreatic ribonuclease A-K7H].
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.
Ljuca
F
F
Zavod za fiziologiju, Medicinski fakultet Tuzla Univerziteta u Tuzli.
Moussaoui
M
M
Boix
E
E
Nogues
V
V
Ljuca
D
D
Cuchillo
C M
CM
D002384:Catalysis; D011994:Recombinant Proteins / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*
D011994:Recombinant Proteins; D012259:Ribonuclease, Pancreatic
Kataliticke osobine rekombinantne pankreasne ribonukleaze A-K7H.