Kinetic studies of guanine recognition and a phosphate group subsite on ribonuclease T1 using substitution mutants at Glu46 and Lys41.
pH-Dependent kinetic studies were performed with ribonuclease T1 (RNase T1) and its Glu46Ser, Lys41Met, and Lys41Thr mutants with GpC and polyinosinic acid (PolyI) as substrates. Plots of pH versus log(k(cat)/K(M)) for both substrates had ascending slopes that were significantly greater for RNase T1 compared with Glu46Ser-RNase T1, which indicated that the gamma-carboxyl group of conserved Glu46 must be deprotonated (anionic) for maximal interaction with N1H and N2H of the guanine moiety of GpC or the N1H of the hypoxanthine moiety of PolyI. The involvement of the epsilon -ammonium group of nonconserved Lys41 at the 2p subsite (i.e., for an RNA phosphate group two nucleotide positions 5'-upstream from the active site) was supported by comparisons of Lys41Met-RNase T1 and Lys41Thr-RNase T1 with wild-type. These mutants shared identical catalytic properties (i.e., k(cat) and K(M)) with wild-type using GpC as a substrate. However, k(cat)/K(M) for both were identical with each other but lower than those for wild-type when PolyI was the substrate (PolyI has a phosphate group that could interact at a putative 2p site). The pH dependence of this latter difference can be interpreted as reflecting the loss of the 2p subsite interaction with the wild-type enzyme upon deprotonation of the epsilon -ammonium group of Lys41. Subsite interactions for ribonucleases are shown to mainly increase k(cat) and result in an attenuated pH dependence of k(cat)/K(M).
Jo Chitester
Betty
B
Department of Chemistry, Kent State University, 44242, Kent, OH, USA
Walz
Frederick G
FG
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D003596:Cytosine; D017931:DNA Primers; D015226:Dinucleoside Phosphates / Q000378:metabolism; D005973:Glutamine; D006147:Guanine; D007700:Kinetics; D008239:Lysine; D008715:Methionine; D016297:Mutagenesis, Site-Directed; D010710:Phosphates; D011069:Poly I / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D006163:Ribonuclease T1 / Q000737:chemistry* / Q000378:metabolism*; D012694:Serine; D013379:Substrate Specificity
D017931:DNA Primers; D015226:Dinucleoside Phosphates; D010710:Phosphates; D011994:Recombinant Proteins; D005973:Glutamine; D011069:Poly I; D012694:Serine; D006147:Guanine; D003596:Cytosine; D008715:Methionine; D006163:Ribonuclease T1; D008239:Lysine