Crystallographic Study of Glu58Ala RNase T1.cntdot.2'-Guanosine Monophosphate at 1.9-.ANG. Resolution

Pletinckx, Jurgen; Steyaert, Jan; Zegers, Ingrid; Choe, Huhn; Heinemann, Udo; Wyns, Lode

doi:10.1021/bi00173a006

Cited by 13 publications

(7 citation statements)

References 30 publications

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“…The present study shows that, although a large number of water molecules is not conserved according to the criteria used, a set of conserved water molecules, inherent to the structure of RNase A, has been identified (see Kinemage 1). Such sets of "structural" water molecules were previously observed for RNase TI (Malin et al, 1991;Pletinckx et al, 1994), and for legume lectins (Loris et al, 1994), and seem to be a general feature of protein structures.…”

Section: Conserved Water Sitessupporting

confidence: 58%

The structures of rnase a complexed with 3′‐CMP and d(CpA): Active site conformation and conserved water molecules

et al. 1994

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The interactions of RNase A with cytidine 3'-monophosphate (3"CMP) and deoxycytidyl-3',5'-deoxyadenosine (d(CpA)) were analyzed by X-ray crystallography. The 3'-CMP complex and the native structure were determined from trigonal crystals, and the d(CpA) complex from monoclinic crystals. The differences between the overall structures are concentrated in loop regions and are relatively small. The protein-inhibitor contacts are interpreted in terms of the catalytic mechanism. The general base His 12 interacts with the 2' oxygen, as does the electrostatic catalyst Lys 41. The general acid His 119 has 2 conformations (A and B) in the native structure and is found in, respectively, the A and the B conformation in the d(CpA) and the 3'-CMP complex. From the present structures and from a comparison with RNase T1, we propose that His 119 is active in the A conformation. The structure of the d(CpA) complex permits a detailed analysis of the downstream binding site, which includes His 119 and Asn 71. The comparison of the present RNase A structures with an inhibitor complex of RNase T1 shows that there are important similarities in the active sites of these 2 enzymes, despite the absence of any sequence homology. The water molecules were analyzed in order to identify conserved water sites. Seventeen water sites were found to be conserved in RNase A structures from 5 different space groups. It is proposed that 7 of those water molecules play a role in the binding of the N-terminal helix to the rest of the protein and in the stabilization of the active site.

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Section: Conserved Water Sitessupporting

confidence: 58%

The structures of rnase a complexed with 3′‐CMP and d(CpA): Active site conformation and conserved water molecules

et al. 1994

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“…The same Na + binding site is also observed in the crystal structure of Glu58Ala RNase T1‚2′-GMP (Pletinckx et al, 1994). A similar sodium site of Na107 has not been found in any of other RNase T1 structures.…”

Section: Resultssupporting

confidence: 71%

Crystal Structure of Ribonuclease T1 Carboxymethylated at Glu58 in Complex with 2‘-GMP

et al. 1996

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The carboxymethylation of RNase T1 at the gamma-carboxyl group of Glu58 leads to a complete loss of the enzymatic activity while it retains substrate-binding ability. Accompanying the carboxymethylation, RNase T1 undergoes a remarkable thermal stabilization of 9 degrees C in the melting temperature (Tm). In order to clarify the inactivation and stabilization mechanisms of RNase T1 by carboxymethylation, the crystal structure of carboxymethylated RNase T1 (CM-RNase T1) complexed with 2'-GMP was determined at 1.8 A resolution. The structure, including 79 water molecules and two Na+, was refined to an R factor of 0.194 with 10 354 reflections > 1 sigma (F). The carboxyl group of CM-Glu58, which locates in the active site, occupies almost the same position as the phosphate group of 2'-GMP in the crystal structure of intact RNase T1.2'-GMP complex. Therefore, the phosphate group of 2'-GMP cannot locate in the active site but protrudes toward the solvent. This forces 2'-GMP to adopt an anti form, which contrasts with the syn form in the crystal of the intact RNase T1.2'-GMP complex. The inaccessibility of the phosphate group to the active site can account for the lack of the enzymatic activity in CM-RNase T1. One of the carboxyl oxygen atoms of CM-Glu58 forms two hydrogen bonds with the side-chains of Tyr38 and His40. These hydrogen bonds are considered to mainly contribute to the higher thermal stability of CM-RNase T1. Another carboxyl oxygen atoms of CM-Glu58 is situated nearby His40 and Arg77. This may provide additional electrostatic stabilization.

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“…It has been shown however, that the unprotonated His40 takes over the role of general base if Glu58 is replaced by alanine [18]. This mutant uses two histidines as acid^base pair and may well function according to a more classical mechanism, as is the case for RNase A. Alternatively, His40 may abstract the proton from the 2P-OH group via a water molecule that is found in the cavity created by the Glu58Ala mutation [39]. This water molecule may also interact catalytically with the pro-S P oxygen, thus compensating largely for the Glu58Ala mutation.…”

Section: Resultsmentioning

confidence: 99%

Mechanism of RNase T1: concerted triester-like phosphoryl transfer via a catalytic three-centered hydrogen bond

Loverix

Winqvist

Strömberg

et al. 2000

Chemistry & Biology

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Based on these results, we put forward a new triester-like mechanism for the RNase T1 catalyzed reaction that involves a three-centered hydrogen bond between the 2'-OH group, the nonbridging pro-S(P) oxygen and one of the carboxylate oxygens of Glu58. This interaction allows nucleophilic attack on an activated phosphate to occur simultaneously with general base catalysis, ensuring concerted phosphoryl transfer via a triester-like mechanism.

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Crystallographic Study of Glu58Ala RNase T1.cntdot.2'-Guanosine Monophosphate at 1.9-.ANG. Resolution

Cited by 13 publications

References 30 publications

The structures of rnase a complexed with 3′‐CMP and d(CpA): Active site conformation and conserved water molecules

The structures of rnase a complexed with 3′‐CMP and d(CpA): Active site conformation and conserved water molecules

Crystal Structure of Ribonuclease T1 Carboxymethylated at Glu58 in Complex with 2‘-GMP

Mechanism of RNase T1: concerted triester-like phosphoryl transfer via a catalytic three-centered hydrogen bond

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