Ribonuclease A mutant His119 Asn: the role of histidine in catalysis.
Bovine pancreatic ribonuclease A (RNase A) has been widely used as a convenient model for structural and functional studies. The enzyme catalyzes cleavage of phosphodiester bonds in RNA and related substrates. Three amino acid residues located at the active site of RNase A (His12, His119, and Lys41) are known to be involved in catalysis. Mutation of His119 to asparagine was generated to study the role of His119 in RNase A catalysis. The mutant enzyme has been isolated and characterized. The mutation significantly decreases the rate of the transesterification reaction and has no effect on substrate affinity of the enzyme. An analysis of the enzymatic properties of H119N RNase A suggests that the imidazole ring of His119 of the wild-type enzyme must be protonated in an enzyme-substrate productive complex. Thus our results indicate that a contribution of protonated His119 into the catalysis is not restricted to protonation of oxygen atom of the substrate leaving group and that His119 participates directly in a transition state stabilization via hydrogen bonding.
Panov
K I
KI
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow.
Kolbanovskaya
E Y
EY
Okorokov
A L
AL
Panova
T B
TB
Terwisscha van Scheltinga
A C
AC
Karpeisky MYa
Beintema
J J
JJ
D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D011994:Recombinant Proteins; D006639:Histidine; D001216:Asparagine; D012259:Ribonuclease, Pancreatic