Active site of RNase: neutron diffraction study of a complex with uridine vanadate, a transition-state analog.

Abstract

A complex of RNase A with a transition-state analog, uridine vanadate, has been studied by a combination of neutron and x-ray diffraction. The vanadium atom occupies the center of a distorted trigonal bipyramid, with the ribose oxygen 02' at the apical position. Contrary to expectations based on the straightforward interpretation of the known in-line mechanism of action of RNase, nitrogen NE2 of histidine-12 was found to form a hydrogen bond to the equatorial oxygen 08, while nitrogen NZ of lysine41 makes a clear hydrogen bond to the apical oxygen 02'. Nitrogen ND1 of histidine-119 appears to be within a hydrogenbond distance of the other apical oxygen, 07. Two other hydrogen bonds between the vanadate and the protein are made by nitrogen NE2 of glutamine-il and by the amide nitrogen of phenylalanine-120. The observed geometry of the complex may necessitate reinterpretation of the mechanism of action of RNase.Although a number of crystallographic investigations have elucidated the three-dimensional structure of RNase A (1-4) and its proteolytic modification RNase S (5-7), many details of the mechanism of action of this enzyme still remain to be conclusively determined. RNase hydrolyzes single-stranded RNA and requires a pyrimidine base on the 3' side of the cleaved phosphodiester linkage (Fig. 1). The stereochemistry of catalysis was investigated by Usher et aL (8,9), and these experiments have shown that both steps of the reaction proceed through an in-line mechanism. Although Usher did not identify the groups in the active site of the protein responsible for catalysis, these may be inferred from the published structures of RNase and of the complexes with inhibitors (6, 7, 10-12). In the first step of catalysis, histidine-12 removes a proton from the 2' oxygen of the pyrimidine ribose, making the oxygen nucleophilic and capable of attacking the phosphorus. In the transition state, the phosphorus becomes pentacoordinated, with the 2' and 5' oxygens assuming apical positions. The bond between the 5' oxygen and the ribose is cleaved, and the result of the first step of the reaction is a 2',3'-cyclic pyrimidine nucleotide. The second step of the reaction is initiated by an attack of a water molecule activated by histidine-119 on the phosphorus opposite the 2' oxygen, followed by formation of a pentacoordinated intermediate; finally, oxygen 02' is protonated by histidine-12, and a 3' nucleotide is formed. According to this interpretation, nitrogen NE2 of histidine-12 should be in the proximity of the 2' oxygen and nitrogen ND1 of histidine-119 should be near the 5' oxygen in order to fulfill their catalytic roles (13)(14)(15). In addition, because it has been known that lysine-41 is necessary for the catalysis and is present in the active site, this amino acid was usually placed adjacent to one of the other oxygens of the phosphate. Such a model was recently used in the design of an artificial enzyme, which showed limited RNase-like activity (16). Lindquist et al. (17) postulated that a complex of uridine ...

Neutron diffraction studies of Escherichia coli dihydrofolate reductase complexed with methotrexate

“…Nearly all of the D 2 Os that possess the highest B factors are at the extremity of the primary solvent layer, 4-5 Å from the nearest protein atom. We also observe many D 2 Os that form H-bonds with fully exchanged amides, possibly promoting H/D exchange.…”

“…Knowledge of hydrogen positions is crucial for understanding enzyme catalysis and molecular recognition and aids drug design. Neutron crystallography (NC) has set several benchmarks, directly revealing hydrogens in proteins and elucidating enzymatic mechanisms ranging from serine proteases (1) to ribonucleases (2) to aspartic proteases (3). Moreover, several other structures have been solved by using NC (4-7).…”

His···Asp Catalytic Dyad of Ribonuclease A: Structure and Function of the Wild-Type, D121N, and D121A Enzymes

“…In addition, N ζ of Lys41 has been shown to form a hydrogen bond with the 2′-oxygen of 3′-CMP and to a bridging vanadyl oxygen of uridine 2′,3′-cyclic vanadate (58,49). In the D121N and D121A variants, the sidechain of Lys41 has clearly defined density and exists in a fully extended conformation.…”

“…Site-directed mutagenesis studies in which Gln11 is replaced by an alanine, asparagine, and histidine residue have shown that the role of Gln11 is to prevent the nonproductive binding of substrate (41). In the structure of the RNase A•uridine 2′,3′-cyclic vanadate complex, N ε2 of Gln11 forms a hydrogen bond with a nonbridging vanadyl oxygen, perhaps mimicking its interaction with the transition state (58,60). The sidechain N ε2 of Gln11 forms a hydrogen bond with a bridging phosphoryl oxygen in an RNase A•d(CpA) complex, and with a water molecule in an RNase A•3′-CMP complex (49).…”

Shared traits on the reaction coordinates of ribonuclease and an RNA enzyme

“…The relevance of vanadiumoxide binding in the ribonuclease (RNase) A active site [10,12] has been challenged, since amino acids poised for transition-state stabilization did not support roles ascribed by biochemical and kinetic analyses [13]. Complementary studies of RNases have been conducted in the presence of substrate analogs comprising 3′-OH, 2′,5′-linkages at the site of cleavage [14][15][16].…”