Ribonuclease A mutant His119Asn: the role of histidine in catalysis

FEBS Letters

Karpeisky, Marat Ya.¹  Panov, Konstantin I.¹  Terwisscha van Scheltinga, Anke C.²  Kolbanovskaya, Elena Yu.¹  Okorokov, Andrei L.¹  Panova, Tatiana B.¹  Beintema, Jaap J.² 

[1] Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilovstr. 32, Moscow 117984, Russia;Departments of Biochemistry and Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands

关键词: Ribonuclease;    RNase A;    Site-directed mutagenesis;   

DOI  :  10.1016/S0014-5793(96)01173-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown