Protonation States Of Active Site Residues Research Articles

Complementarity of neutron, XFEL and synchrotron crystallography for defining the structures of metalloenzymes at room temperature.

Room-temperature macromolecular crystallography allows protein structures tobe determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B.

QM/MM Study of the Catalytic Reaction of Myrosinase; Importance of Assigning Proper Protonation States of Active-Site Residues.

Myrosinase from Sinapis alba hydrolyzes glycosidic bonds of β-d-S-glucosides. The enzyme shows an enhanced activity in the presence of l-ascorbic acid. In this work, we employed combined quantum mechanical and molecular mechanical (QM/MM) calculations and molecular dynamics simulations to study the catalytic reaction of wild-type myrosinase and its E464A, Q187A, and Q187E mutants. Test calculations show that a proper QM region to study the myrosinase reaction must contain the whole substrate, models of Gln-187, Glu-409, Gln-39, His-141, Asn-186, Tyr-330, Glu-464, Arg-259, and a water molecule. Furthermore, to make the deglycosylation step possible, Arg-259 must be charged, Glu-464 must be protonated on OE2, and His-141 must be protonated on the NE2 atom. The results indicate that assigning proper protonation states of the residues is more important than the size of the model QM system. Our model reproduces the anomeric retaining characteristic of myrosinase and also reproduces the experimental fact that ascorbate increases the rate of the reaction. A water molecule in the active site, positioned by Gln-187, helps the aglycon moiety of the substrate to stabilize the buildup of negative charge during the glycosylation reaction and this in turn makes the moiety a better leaving group. The water molecule also lowers the glycosylation barrier by ∼9 kcal/mol. The results indicate that the Q187E and E464A mutants but not the Q187A mutant can perform the glycosylation step. However, the energy profiles for the deglycosylation step of the mutants are not similar to that of the wild-type enzyme. The Glu-464 residue lowers the barriers of the glycosylation and deglycosylation steps. The ascorbate ion can act as a general base in the reaction of the wild-type enzyme only if the Glu-464 and His-141 residues are properly protonated.

Revised mechanism of carboxylation of ribulose-1,5-biphosphate by rubisco from large scale quantum chemical calculations.

Here, we describe a computational approach for studying enzymes that catalyze complex multi-step reactions and apply it to Ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco), the enzyme that fixes atmospheric carbon dioxide within photosynthesis. In the 5-step carboxylase reaction, the substrate Ribulose-1,5-bisphosphate (RuBP) first binds Rubisco and undergoes enolization before binding the second substrate, CO2 . Hydration of the RuBP.CO2 complex is followed by CC bond scission and stereospecific protonation. However, details of the roles and protonation states of active-site residues, and sources of protons and water, remain highly speculative. Large-scale computations on active-site models provide a means to better understand this complex chemical mechanism. The computational protocol comprises a combination of hybrid semi-empirical quantum mechanics and molecular mechanics within constrained molecular dynamics simulations, together with constrained gradient minimization calculations using density functional theory. Alternative pathways for hydration of the RuBP.CO2 complex and associated active-site protonation networks and proton and water sources were investigated. The main findings from analysis of the resulting energetics advocate major revision to existing mechanisms such that: hydration takes place anti to the CO2 ; both hydration and CC bond scission require early protonation of CO2 in the RuBP.CO2 complex; CC bond scission and stereospecific protonation reactions are concerted and, effectively, there is only one stable intermediate, the C3-gemdiolate complex. Our main conclusions for interpreting enzyme kinetic results are that the gemdiolate may represent the elusive Michaelis-Menten-like complex corresponding to the empirical Km (=Kc ) with turnover to product via bond scission concerted with stereospecific protonation consistent with the observed catalytic rate. © 2018 Wiley Periodicals, Inc.

On the Influence of the Protonation States of Active Site Residues on AChE Reactivation: A QM/MM Approach.

Acetylcholinesterase (AChE), an enzyme of the serine hydrolase superfamily, is a mediator of signal transmission at cholinergic synapses by catalyzing acetylcholine cleavage into acetate and choline. This enzyme is vulnerable to covalent inhibition by organophosphate compounds (like VX). Covalent inhibition of AChE does not revert spontaneously. Known reactivator compounds have limited action in restoring catalytic activity. QM/MM simulations of VX-inhibited AChE reactivation by pralidoxime (2-PAM), a classical reactivator, were performed. These afforded a broad view of the effect of protonation states of active-site residues, and provide evidence for the role of Glu202, which needs to be protonated for reactivation to occur. In situ deprotonation of 2-PAM for both protonation states of Glu202 showed that His447 is able to deprotonate 2-PAM with the assistance of Glu202. Because the active site of serine hydrolases is highly conserved, this work provides new insights on the interplay between the catalytic triad residues and this glutamate, newly identified as protonatable.

Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate.

ABSTRACTThe monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M β-lactamase but not for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A β-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, as previously proposed.