Tripeptides comprising amino acids with neutral side chains interact with alkaline earth metal ions to form gas-phase anionic complexes of the composition [tripept + Met2"1" -3H+]" under fast atom bombardment. The metal ion binds to the deprotonated C-terminal carboxylate group and to the two amide nitrogens. Because the C-terminal and the central amino acid are tightly bound by the metal ion, they are not vulnerable to collisionally activated decompositions in a tandem mass spectrometer. Instead, the significant fragmentations occur at the N-terminal amino acid site, which is the least tightly bonded. Ions are formed by elimination of an imine and the imine plus CO from the N-terminus (product ions are assigned as x2 + H and y2, respectively). Other major fragmentations of this complex include dehydrogenation and loss of an ammonia molecule. Peptides with functionalized side chains, such as those of serine, threonine, and phenylalanine, lose the side chains readily when they are bound to metal ions and submitted to collisional activation. Other fragmentation channels are largely suppressed, indicating direct metal ion-side chain interaction. Fragmentation mechanisms are proposed on the basis of results with isotopically labeled peptides and from MS/MS/MS experiments.
The X-ray crystal structure of a uridine vanadate-ribonuclease A complex has been determined at 1.3 A resolution. The resulting structure includes all 124 amino-acid residues, a uridine vanadate, 131 water molecules, and a single bound 2-methyl-2-propanol. Side chains of 11 surface residues showing discrete disorder were modeled with multiple conformations. The final crystallographic R factor is 0.197. Structures obtained from high-level ab initio quantum calculations of model anionic oxyvanadate compounds were used to probe the effects of starting structure on the refinement process and final structure of the penta-coordinate phosphorane analog, uridine vanadate. The least-squares refinement procedure gave rise to the same final structure of the inhibitor despite significantly different starting models. Comparison with the previously determined complex of ribonuclease A with uridine vanadate obtained from a joint X-ray/neutron analysis (6RSA) [Wlodawer, Miller & Sjölin (1983). Proc. Natl Acad. Sci. USA, 80, 3628-3631] reveals similarities in the overall enzyme structure and the relative position of the key active-site residues, Hisl2, His119 and Lys41, but significant differences in the V-O bond distances and angles. The influence of ligand binding on the enzyme structure is assessed by a comparison of the current X-ray structure with the phosphate-free ribonuclease A structure (7RSA) [Wlodawer, Svensson, Sjölin & Gilliland (1988). Biochemistry, 27, 2705-2717]. Ligand binding alters the solvent structure, distribution and number of residues with multiple conformations, and temperature factors of the protein atoms. In fact, the temperature factors of atoms of several residues that interact with the ligand are reduced, but those of the atoms of several residues remote from the active site exhibit marked increases.
The transphosphorylation step in the enzyme-catalyzed hydrolysis of phosphate esters by Ribonuclease A (RNase A) is explored using ab initio quantum chemical methods. For the first time, components found in the RNase A active site are included in the all-electron chemical model, made up of 2-hydroxyethyl methyl phosphate monoanion used as the substrate, and small model compounds used to mimic the three important residues, His-12, His-119, and Lys-41, found in the RNase A active site. The remainder of the immediate active site, including ten residues and six bound water molecules, is treated using effective fragment potentials (EFPs) incorporated directly into the Hamiltonian of the quantum system. The EFPs, derived from separate quantum calculations on individual components, are constructed to accurately represent the correct electrostatics and polarization fields of each component. High-resolution X-ray crystallographic data are used to assign the fixed relative positions of each component in the quantum and EFP regions. Characterization of the salient stationary points along the transphosphorylation reaction pathway at the RHF level using a 3-21+G(d) basis set reveals several low-barrier proton transfer steps between the substrate and the active site residues which allow transphosphorylation to occur with modest activation, consistent with the experimental data. Mpller-Plesset perturbation theory (MP2) and density functional theory methods utilizing a larger 6-31+G(d) basis are also used to explore the effects of electron correlation on the surface energetics. Consistent with expectations, the electrostatic field effects from the EFPs used to represent the non-participating parts of the active site are found to differentially stabilize certain structures along the reaction pathway.
A joint X-ray crystallographic (1.3 Å resolution) and ab initio quantum mechanical analysis of a uridine vanadate−ribonuclease A complex (UV−RNase A) is undertaken to probe specific aspects of the microscopic mechanism by which ribonuclease functions to catalyze the hydrolysis of its natural substrate, phosphate esters. Comparison of the structural features of the vanadate portion from the final X-ray refinement with the oxy-vanadate model compounds determined computationally provides direct evidence of the likely protonation state of the UV inhibitor bound in the active site. Specifically, the UV bound in the active site of UV−RNase A is found to be monoanionic, and the most likely source of this proton is from the active site residue His12. Together with the structural data, these results strongly suggest that even though His12 may act as the catalytic base in the first step of the mechanism, transphosphorylation, and the catalytic acid in the second step, hydrolysis, it must also play an additional, although perhaps secondary, role in stabilizing the pentacoordinate phosphorane structure through proton transfer. On the basis of its close proximity to critical vanadate oxygen in the UV, and data obtained from a previous computational study, Lys41 is likely to play a more intimate role in the catalytic mechanism than previously proposed, potentially acting as the catalytic base in certain cases. Two possible detailed microscopic mechanisms are presented.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.