It has been established that Tyr-42, Tyr-45, and Glu-46 take part in a structural motif that renders guanine specificity to ribonuclease T1. We report on the impact of Tyr-42, Tyr-45, and Glu-46 substitutions on the guanine specificity of RNase T1. The Y42A and E46A mutations profoundly affect substrate binding. No such effect is observed for Y45A RNase T1. From the kinetics of the Y42A/Y45A and Y42A/E46A double mutants, we conclude that these pairs of residues contribute to guanine specificity in a mutually independent way. From our results, it appears that the energetic contribution of aromatic face-to-face stacking interactions may be significant if polycyclic molecules, such as guanine, are involved.The energetics of virtually all binding properties in proteins is the culmination of a complex set of intermolecular interactions. The individual contributions and the mutual interdependence of these interactions are currently being probed through protein engineering by many research groups. The most powerful experimental approach is to analyze the effects of single mutations on binding, turnover, or conformational stability and to compare these with the properties of proteins where multiple mutations are combined in one molecule (1-4).The functional role and cooperative interplay between catalytic residues have been investigated in detail for a number of enzymes including subtilisin (5, 6), staphylococcal nuclease (7,8), and ribonuclease T1 (9). In each case, the free energy barriers to substrate turnover introduced by mutations of catalytic residues are not additive in the corresponding double (or multiple) mutants. Residues involved in the process of breaking and forming covalent bonds appear to contribute to turnover in a mutually dependent way. Far less information is available on the additivity of molecular interactions involved in substrate binding. In this study, we investigate the mutual dependence of interactions at the guanine-binding site of ribonuclease T1 by protein engineering.Ribonuclease T1 (RNase T1; EC 3.1.27.3) of the slime mold Aspergillus oryzae (10) is the best known representative of a large family of homologous microbial ribonucleases with members in the prokaryotic and eukaryotic worlds (11,12). RNase T1 has a pronounced specificity for the base guanine; kinetic studies on the trans-esterification of dinucleoside phosphates revealed that the specificity constant (k cat /K m ) for GpN 1 substrates is ϳ10 6 -fold greater than for corresponding ApN substrates and at least 10 8 -fold greater than for CpN and UpN substrates (13). The three-dimensional structure of RNase T1 complexed with the competitive inhibitor 2Ј-GMP (14, 15) provides a structural basis for understanding the enzyme's specificity. In the complex, the hydrogen-bonding potential of the guanine base is completely saturated by complementary donor/ acceptor sites on the enzyme involving the backbone atoms of Asn-43, Asn-44, and Asn-98 and the side chain carboxyl group of Glu-46 (Fig. 1). The N(1)-H-Glu-46 O-⑀1, N(2)-H-Glu-46 O-⑀...

The function of the conserved Phe 100 residue of RNase TI (EC 3.1.27.3) has been investigated by site-directed mutagenesis and X-ray crystallography. Replacement of Phe 100 by alanine results in a mutant enzyme with kc,, reduced 75-fold and a small increase in K , for the dinucleoside phosphate substrate GpC. The Phe 100 Ala substitution has similar effects on the turnover rates of GpC and its minimal analogue GpOMe, in which the leaving cytidine is replaced by methanol. The contribution to catalysis is independent of the nature of the leaving group, indicating that Phe 100 belongs to the primary site. The contribution of Phe 100 to catalysis may result from a direct van der Waals contact between its aromatic ring and the phosphate moiety of the substrate. Phe 100 may also contribute to the positioning of the pentacovalent phosphorus of the transition state, relative to other catalytic residues. If compared to the corresponding wild-type data, the structural implications of the mutation in the present crystal structure of Phe 100 Ala RNase T, complexed with the specific inhibitor 2'-GMP are restricted to the active site. Repositioning of 2'-GMP, caused by the Phe 100 Ala mutation, generates new or improved contacts of the phosphate moiety with Arg 77 and His 92. In contrast, interactions with the Glu 58 carboxylate appear to be weakened. The effects of the His 92 Gln and Phe 100 Ala mutations on GpC turnover are additive in the corresponding double mutant, indicating that the contribution of Phe 100 to catalysis is independent of the catalytic acid His 92. The present results lead to the conclusion that apolar residues may contribute considerably to catalyze conversions of charged molecules to charged products, involving even more polar transition states.

The reoccurrence of water molecules in crystal structures of RNase T1 was investigated. Five waters were found to be invariant in RNase T1 as well as in six other related fungal RNases. The structural, dynamical, and functional characteristics of one of these conserved hydration sites~WAT1! were analyzed by protein engineering, X-ray crystallography, and 17 O and 2 H nuclear magnetic relaxation dispersion~NMRD!. The position of WAT1 and its surrounding hydrogen bond network are unaffected by deletions of two neighboring side chains. In the mutant Thr93Gln, the Gln93NE2 nitrogen replaces WAT1 and participates in a similar hydrogen bond network involving Cys6, Asn9, Asp76, and Thr91. The ability of WAT1 to form four hydrogen bonds may explain why evolution has preserved a water molecule, rather than a side-chain atom, at the center of this intricate hydrogen bond network. Comparison of the 17 O NMRD profiles from wild-type and Thr93Gln RNase T1 yield a mean residence time of 7 ns at 27 8C and an orientational order parameter of 0.45. The effects of mutations around WAT1 on the kinetic parameters of RNase T1 are small but significant and probably relate to the dynamics of the active site.Keywords: conserved water; hydrogen bond network; NMRD; protein hydration; residence time; RNase T1The structure and function of proteins and other biological macromolecules depend on their interaction with the surrounding aqueous solvent via the hydrophobic effect, dielectric screening, and specific hydrogen bonds. Most globular proteins contain a few water molecules buried in cavities. Such internal water molecules heal packing defects and extend the intramolecular hydrogen bond framework, thus contributing importantly to protein structure and stability, and are sometimes directly involved in enzyme catalysis Meyer, 1992!. Because they are conserved among homologous proteins to the same extent as amino acid residues~Baker, 1995!, internal water molecules are clearly an integral part of the protein structure. The residence times of these internal water molecules are typically in the range 10 Ϫ8 -10 Ϫ6 s at ambient temperature~Den-isov & Halle, 1996!, but may be as long as 200 ms at 27 8C Denisov et al., 1996!.The several hundred water molecules that make contact with the surface of a small protein are considered much more mobile~Den-isov & Halle, 1996!, and the relationship between hydration in solution and the water sites that are found in macromolecular crystal structures is poorly understood. In the present study, we analyze the reoccurrence of water molecules in the different crystal structures of RNase T1 and the related RNases F1, Ms, Ap, Pb, Th, and U2. Based on the results of this comparative study, a single water molecule is chosen~referred to as WAT1!, of which the structural, dynamical, and functional properties are investigated using site-directed mutagenesis and X-ray crystallography. From the analysis of NMRD profiles, the mean residence time and orientational order parameter of WAT1 are calculated. This is t...