Abstract
The complex formed between the enzyme ribonuclease T1 (EC 3.1.27.3) and its specific inhibitor 2'-guanylic acid (2'-GMP) has been refined to R = 0.180 using x-ray diffraction data to 1.9-A resolution. The protein molecule displays a compact fold; a 4.5 turn alpha-helix packed over an antiparallel beta-pleated sheet shields most of the hydrophobic interior of the protein against the solvent. The extended pleated sheet structure of ribonuclease T1 is composed of three long and four short strands building up a two-stranded minor beta-sheet near the amino terminus and a five-stranded major sheet in the interior of the protein molecule. In the complex with ribonuclease T1, the inhibitor 2'-guanylic acid adopts the syn-conformation and C2'-endo sugar pucker. Binding of the nucleotide is mainly achieved through amino acid residues 38-46 of the protein. The catalytically active amino acid residues of ribonuclease T1 (His40, Glu58, Arg77, and His92) are located within the major beta-sheet which, as evident from the analysis of atomic temperature factors, provides an environment of minimal local mobility. The geometry of the active site is consistent with a mechanism for phosphodiester hydrolysis where, in the transesterification step, His40 and/or Glu58 act as a general base toward the ribose 2'-hydroxyl group and His92, as a general acid, donates a proton to the leaving 5'-hydroxyl group.
Highlights
The complex formed between the enzyme ribonuclef-amily spans the divide between the prokaryotic and eukaryase TI (EC 3.1.27.3) and its specific inhibitor2”guanylic acid (2’-GMP) has been rqfinedRt=o 0.180 using otic kingdoms
The bound inhibitor of RNase TI, 2'-GMP, the polypeptide backbone, and theamino acid side chains are drawn with decreasing line width
TI.The most detailed information comes from NMR experiments: One-dimensional nuclear Overhauser effect (NOE)
Summary
Protein Conformation-The r.m.s. distance between equivalentmainchainatoms .Of the refined andtheunrefined RNase TI models is 1.3 A. 0 hydrogen bonds connecting different polar residues in the N-terminal half of the helix further strands in the pleated sheets of RNase TI, thfeollowingmean stabilizes the structureby side chain-backbone and side chainside chain hydrogen bonds. While Alax7has adopted backbone torsionangles -97"/-38" of hydrogen bonds is observed, with thepossible exception of close to the a-helicarlegion of conformational space, all other protein side chain-side chain bonds that show-an unusually amino acid residues involved in the bulge structure display small average donor-acceptor distanceof 2.77 A. Linked to Val7' of Ps by two hydrogen bonds This portion of Local Flexibility-Since the mobility of amino acid residues the major sheet is ratherrigid as jydged by a mean crystallo- in crystalline proteins is restricted by lattice contacts, in the graphic temperature factor of 7.2 A' for the backbone atoms thermal factorhistogram (Fig. 8) those amino acid side chain of thetripeptide G1u"-Trp"-Pro. Most of the side chain of Trpsg showsvery limited-mobility as evident them do not lie within the helix and sheet regions of the from a mean temperature factor of6.0 42, which is signifi- protein and adopt intermediate temperature factors, indicatcantly below the average B value of 12.9 A* for all side chain ing that intermolecular contacts do not determine the segatoms of the proteinmolecule
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