Role of aspartic acid 121 in human pancreatic ribonuclease catalysis.
Bovine pancreatic ribonuclease (RNase A) is one of the most well studied enzymes of the ribonuclease family, unlike its human counterpart, the human pancreatic ribonuclease (HPR), whose physiological role in the body is not clearly understood. Human pancreatic ribonuclease consists of 128 amino acids and the main residues located in the active site of RNase A are also conserved in HPR. In the current study, to investigate the role of Asp-121 in the catalytic activity of human pancreatic ribonuclease, several variants were generated in which Asp-121 was either mutated to an alanine or C-terminal residues beyond Asp-121, and Phe-120 were deleted. The HPR mutants were cloned, expressed in E. coli and purified to homogeneity, and functionally characterized. The mutation D121A in HPR significantly decreased the rate of the enzymatic reaction, however this decrease was not universally observed for all substrates studied. Removal of the seven C-terminal amino acid residues thereby exposing Asp-121 yielded an HPR mutant with enhanced activity, however a further deletion removing Asp-121 resulted in the complete inactivation of HPR. Our results indicate that Asp-121 is crucial for the catalytic activity of HPR and may be involved in the depolymerization activity of the enzyme.
Gaur
Deepak
D
Immunochemistry Laboratory, National Institute of Immunology, New Delhi, India.
Batra
Janendra K
JK
D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D011994:Recombinant Proteins; D001224:Aspartic Acid; D010649:Phenylalanine; D012259:Ribonuclease, Pancreatic; D000409:Alanine