Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.3 (RNase T1)
 1,228 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We used harmonic-content frequency-domain fluorometry to determine the anisotropy decays of a variety of single tryptophan peptides and proteins. Resolution of the rapid and complex anisotropy decays was enhanced by global analysis of the data measured in the presence of quenching by either oxygen or acrylamide. For each protein, and for each quencher, data were obtained at four to six quencher concentrations, and the data analyzed globally to recover the anisotropy decay. The decrease in decay times produced by quenching allows measurements to an upper frequency limit of 2 GHz. The chosen proteins provided a range of exposures of the tryptophan residues to the aqueous phase, these being ACTH, monellin, Staphylococcus nuclease and ribonuclease T1, in order of decreasing exposure. Examination of indole and several small peptides demonstrates the resolution limitations of the measurements; a correlation time of 12 ps was measured for indole in methanol at 40 degrees C. Comparison of the anisotropy decays of gly-trp-gly with leu-trp-leu revealed stearic effects of the larger leucine side chains on the indole ring. The anisotropy decay of gly-trp-gly revealed a 40 ps component for the indole side chain, which was resolved from the overall 150 ps correlation time of the tripeptide. Only the longer correlation time was observed for leu-trp-leu. With the exception of ribonuclease T1, each of the proteins displayed a subnanosecond component in the anisotropy decay which we assign to independent motions of the tryptophan residues. For example, Staphylococcus nuclease and monellin displayed segmental tryptophan motions with correlation times of 80 and 275 ps, respectively. The amplitudes of the rapid components increased with increasing exposure to the aqueous phase. These highly resolved anisotropy decays for proteins of known structure are suitable for comparison with molecular dynamic simulations.
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PMID:Anisotropy decays of single tryptophan proteins measured by GHz frequency-domain fluorometry with collisional quenching. 164 47

We previously presented evidence that ribonuclease T1 (RNase T1; EC 3.1.27.3) contains a subsite that, by interacting with the leaving nucleoside N of GpN dinucleoside phosphate substrates, contributes to catalysis. The kcat values for transphosphorylation follow the order GpC greater than GpA greater than GpU whereas the equilibrium dissociation constants for these substrates are very similar [Steyaert, J., Wyns, L., & Stanssens, P. (1991) Biochemistry (preceding paper in this issue)]. Consistent with this notion, we find that the rate of transesterification of the synthetic substrate GpMe, in which the leaving nucleoside is replaced by a methanol group, is at least 3 orders of magnitude lower than that of GpN substrates. The enzyme's affinity for GpMe is very similar to that for the various GpN substrates, indicating that the apparent contribution of the leaving nucleoside to ground-state binding is minimal. To identify the side chains that belong to the RNase T1 subsite, we searched for amino acid substitutions that differentially affect the transesterification kinetics of GpNs versus GpMe. The Asn36Ala, Tyr38Phe, His92Gln, and Asn98Ala mutants have been analyzed. Of these, the Asn36Ala and Asn98Ala substitutions reduce the transphosphorylation rate of the different GpNs considerably whereas they have virtually no effect on the rate of GpMe transphosphorylation. This observation shows that the Asn36 and Asn98 amide functions are part of the RNase T1 subsite. The sum of the contributions of the two residues accounts quite precisely for the differences in turnover rates among GpC, GpA, and GpU.
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PMID:Subsite interactions of ribonuclease T1: Asn36 and Asn98 accelerate GpN transesterification through interactions with the leaving nucleoside N. 165 3

Ribonuclease T1 contains a subsite which by interacting with the leaving nucleoside N of GpN dinucleoside phosphate substrates, contributes to catalysis [Steyaert, J., Wyns, L. & Stanssens, P. (1991) Biochemistry 30, 8661-8665]. The Asn36Ala and Asn98Ala mutations reduce the transesterification rates of GpA, GpC and GpU considerably whereas they have virtually no effect on the transesterification kinetics of the synthetic substrate guanosine 3'-(methyl phosphate) (GpMe) (in which the leaving nucleoside is replaced by methanol), indicating that the Asn36 and Asn98 side chains are part of the RNase T1 subsite [Steyaert, J., Haikal, A. F., Wyns, L. & Stanssens, P. (1991) Biochemistry 30, 8666-8670]. The kinetics of the Asn36Ala, Asn98Ala and wild-type catalyzed transesterification of guanosine 3'-(5'-D-ribosyl phosphate) (GpRib), another GpN analog in which the leaving groups is replaced by D-ribose, enables the mapping of the subsite interactions provided by Asn36 and Asn98. We find that the Asn36 amide function contributes 4.6 kJ/mol to catalysis through interactions with the ribose moiety of the leaving nucleoside. Asn98 is at least in part responsible for the subsite preference for cytidine; the Asn98 side chain preferentially binds cytosine as the leaving nucleoside base.
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PMID:Dissection of the ribonuclease T1 subsite. The transesterification kinetics of Asn36Ala and Asn98Ala ribonuclease T1 for minimal dinucleoside phosphates. 173 39

We report on the functional cooperativity of the primary site and the subsite of ribonuclease T1 (RNase T1; EC 3.1.27.3). The kinetic properties of the single Tyr-38-Phe and Asn-98-Ala mutants have been compared with those of the corresponding double mutant. The Tyr-38-Phe mutation has been used to probe enzyme-substrate interactions at the primary site; the Asn-98-Ala mutation monitors subsite interactions. In addition to the dinucleoside phosphate substrate GpC, we measured the kinetics for GpMe, a synthetic substrate in which the leaving nucleoside cytosine has been replaced by methanol. All data were combined in a triple mutant box to analyze the interplay between Tyr-38, Asn-98, and the leaving group. The free energy barriers to kcat, introduced by the single Tyr-38-Phe and Asn-98-Ala mutations are not additive in the corresponding double mutant. The energetic coupling between both mutations is independent of the binding of the leaving cytosine at the subsite. We conclude that the coupling of the Tyr-38-Phe and Asn-98-Ala mutations arises through distortion or reorientation of the 3'-guanylic acid moiety bound at the primary site. The experimental data indicate that the enzyme-substrate interactions beyond the scissile phosphodiester bond contribute to catalysis through the formation of new or improved contacts in going from ground state to transition state, which are functionally independent of primary site interactions.
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PMID:Investigation of the functional interplay between the primary site and the subsite of RNase T1: kinetic analysis of single and multiple mutants for modified substrates. 820 24

The function of the conserved Phe 100 residue of RNase T1 (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 kcat reduced 75-fold and a small increase in Km 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 T1 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.
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PMID:A catalytic function for the structurally conserved residue Phe 100 of ribonuclease T1. 884 43