Quantitative analysis of the contribution of Glu46 and Asn98 to the guanosine specificity of ribonuclease T1.

TitleQuantitative analysis of the contribution of Glu46 and Asn98 to the guanosine specificity of ribonuclease T1.
Publication TypeJournal Article
Year of Publication1991
AuthorsSteyaert, J., C. Opsomer, L. Wyns, and P. Stanssens
JournalBiochemistry
Volume30
Issue2
Pagination494-9
Date Published1991 Jan 15
ISSN0006-2960
KeywordsAsparagine, Aspergillus oryzae, Binding Sites, DNA Mutational Analysis, Glutamates, Guanine, Guanosine, Hydrogen Bonding, Hypoxanthine, Hypoxanthines, Kinetics, Oligonucleotides, Recombinant Proteins, Ribonuclease T1, Substrate Specificity, Xanthine, Xanthines
Abstract

In the crystal structure of the ribonuclease T1 (RNase T1; EC 3.1.27.3)-2'-GMP complex the hydrogen-bonding potential of the guanine base is saturated [Arni, R., Heinemann, U., Tokuoka, R., & Saenger, W. (1988) J. Biol. Chem. 263, 15358-15368]. The oxygens of the Glu46 carboxylate and the Asn98 main-chain carbonyl act as hydrogen-bond acceptors for the N(1)H-C(2)-N(2)H2 part of the base. We measured the transesterification kinetics of wild-type and Glu46Ala RNase T1 using the GpU, IpU, and XpU series of analogous substrates. We found that the N(1)H---Glu46 O epsilon 1, the N(2)H---Glu46 O epsilon 2, and the N(2)H---Asn98 O hydrogen bonds have an apparent contribution of 2.7, 1.1, and 1.2 kcal/mol to the interaction energy of the enzyme and the transition state of the substrate. Wild-type RNase T1 discriminates guanine from nonionized xanthine (a guanine analogue in which the exocyclic amino group is replaced by an oxygen) by about 4.4 kcal/mol. Loss of the specific hydrogen bonds with the exocyclic amino group of the guanine base accounts for 2.4 kcal/mol of this discrimination energy; 2.0 kcal/mol is due to unfavorable non-H-bonded oxygen-oxygen contacts in the enzyme-xanthine complex. A pH dependence study shows that the deprotonated form of xanthine (i.e., the 6-keto-2-enolate anion; pKa = 5.4) is far less preferred, if not excluded, as substrate by wild-type RNase T1; this may be attributed to an electrostatic repulsion of the negatively charged xanthine by the Glu46 carboxylate group.

Alternate JournalBiochemistry
PubMed ID1899029