Quantitative analysis of the contribution of Glu46 and Asn98 to the guanosine specificity of ribonuclease T1.
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.
Steyaert
J
J
Plant Genetic Systems N.V., Gent, Belgium.
Opsomer
C
C
Wyns
L
L
Stanssens
P
P
D001216:Asparagine; D001236:Aspergillus oryzae / Q000201:enzymology; D001665:Binding Sites; D004252:DNA Mutational Analysis; D005971:Glutamates; D006147:Guanine / Q000378:metabolism; D006151:Guanosine / Q000378:metabolism; D006860:Hydrogen Bonding; D019271:Hypoxanthine; D007042:Hypoxanthines / Q000378:metabolism; D007700:Kinetics; D009841:Oligonucleotides / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D006163:Ribonuclease T1 / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D019820:Xanthine; D014970:Xanthines / Q000378:metabolism
D005971:Glutamates; D007042:Hypoxanthines; D009841:Oligonucleotides; D011994:Recombinant Proteins; D014970:Xanthines; D006151:Guanosine; D019820:Xanthine; D019271:Hypoxanthine; D006147:Guanine; D001216:Asparagine; D006163:Ribonuclease T1