Active-site characterization of S1 nuclease. II. Involvement of histidine in catalysis.
Modification of the histidine residues of purified S1 nuclease resulted in loss of its single-stranded (ss)DNAase, RNAase and phosphomonoesterase activities. Kinetics of inactivation indicated the involvement of a single histidine residue in the catalytic activity of the enzyme. Furthermore, histidine modification was accompanied by the concomitant loss of all the activities of the enzyme, indicating the presence of a common catalytic site responsible for the hydrolysis of ssDNA, RNA and 3'-AMP. Substrate protection was not observed against Methylene Blue- and diethyl pyrocarbonate (DEP)-mediated inactivation. The histidine (DEP)-modified enzyme could effectively bind 5'-AMP, a competitive inhibitor of S1 nuclease, whereas the lysine (2,4,6-trinitrobenzenesulphonic acid)-modified enzyme showed a significant decrease in its ability to bind 5'-AMP. The inability of the substrates to protect the enzyme against DEP-mediated inactivation, coupled with the ability of the modified enzyme to bind 5'-AMP effectively, suggests the involvement of histidine in catalysis.
Gite
S
S
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Reddy
G
G
Shankar
V
V
D001236:Aspergillus oryzae / Q000201:enzymology; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D010777:Photochemistry; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000037:antagonists & inhibitors / Q000737:chemistry*
D006639:Histidine; D015719:Single-Strand Specific DNA and RNA Endonucleases; D004047:Diethyl Pyrocarbonate