Evidence that three histidine residues of a base non-specific and adenylic acid preferential ribonuclease from Rhizopus niveus are involved in the catalytic function.
In order to study the structure-function relationship of an RNase T2 family enzyme, RNase Rh, from Rhizopus niveus, we investigated the roles of three histidine residues by means of site-specific mutagenesis. One of the three histidine residues of RNase RNAP Rh produced in Saccharomyces cerevisiae by recombinant DNA technology was substituted to a phenylalanine or alanine residue. A Phe or Ala mutant enzyme at His46 or His109 showed less than 0.03%, but a mutant enzyme at His104 showed 0.54% of the enzymatic activity of the wild-type enzyme with RNA as a substrate. Similar results were obtained, when ApU was used as a substrate. The binding constant of a Phe mutant enzyme at His46 or His109 towards 2'-AMP decreased twofold, but that at His104 decreased more markedly. Therefore, we assumed that these three histidine residues are components of the active site of RNase Rh, that His104 contributes to some extent to the binding and less to the catalysis, and that the other two histidine residues and one carboxyl group not yet identified are probably involved in the catalysis. We assigned the C-2 proton resonances of His46, His104, and His109 by comparison of the 1H-NMR spectra of the three mutant enzymes containing Phe in place of His with that of the native enzyme, and also determined the individual pKa values for His46 and His104 to be 6.70 and 5.94. His109 was not titrated in a regular way, but the apparent pKa value was estimated to be around 6.3. The fact that addition of 2'-AMP caused a greater effect on the chemical shift of His104 in the 1NMR spectra as compared with those of the other histidine residues, may support the idea described above on the role of His104.
Ohgi
K
K
Department of Microbiology, Hoshi College of Pharmacy, Tokyo.
Horiuchi
H
H
Watanabe
H
H
Iwama
M
M
Takagi
M
M
Irie
M
M
D000241:Adenosine / Q000737:chemistry; D000249:Adenosine Monophosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012233:Rhizopus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship; D013696:Temperature
D005656:Fungal Proteins; D000249:Adenosine Monophosphate; D006639:Histidine; D004722:Endoribonucleases; C075842:ribonuclease Rh; C022543:ribonuclease T(2); D000241:Adenosine