For present purposes, a protein-bound metal site consists of one or more metal ions and all protein side chain and exogenous bridging and terminal ligands that define the first coordination sphere of each metal ion. Such sites can be classified into five basic types with the indicated functions: (1) structural -- configuration (in part) of protein tertiary and/or quaternary structure; (2) storage -- uptake, binding, and release of metals in soluble form: (3) electron transfer -- uptake, release, and storage of electrons; (4) dioxygen binding -- metal-O{sub 2} coordination and decoordination; and (5) catalytic -- substrate binding, activation, and turnover. The authors present here a classification and structure/function analysis of native metal sites based on these functions, where 5 is an extensive class subdivided by the type of reaction catalyzed. Within this purview, coverage of the various site types is extensive, but not exhaustive. The purpose of this exposition is to present examples of all types of sites and to relate, insofar as is currently feasible, the structure and function of selected types. The authors largely confine their considerations to the sites themselves, with due recognition that these site features are coupled to protein structure at all levels. In themore » next section, the coordination chemistry of metalloprotein sites and the unique properties of a protein as a ligand are briefly summarized. Structure/function relationships are systematically explored and tabulations of structurally defined sites presented. Finally, future directions in bioinorganic research in the context of metal site chemistry are considered. 620 refs.« less

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Structural and Functional Aspects of Metal Sites in Biology

The role of the lone pair of electrons of Pb(II) in determining the coordination geometry is analyzed from crystallographic studies and ab initio molecular orbital optimizations. Of particular interest are factors that contribute to the disposition of ligands around the lead with geometries that are (1) holodirected, in which the bonds to ligand atoms are distributed throughout the surface of an encompassing globe, and (2) hemidirected, in which the bonds to ligand atoms are directed throughout only part of an encompassing globe, i.e., there is an identifiable void in the distribution of bonds to the ligands. The preferred coordination numbers for lead were found to be 4 for Pb(IV) and 4 and 6 for Pb(II). All Pb(IV) structures in the CSD have a holodirected coordination geometry. Pb(II) compounds are hemidirected for low coordination numbers (2−5) and holodirected for high coordination numbers (9, 10), but for intermediate coordination numbers (6−8), examples of either type of stereochemistry are found. A...

Lone Pair Functionality in Divalent Lead Compounds

Molecular and industrial aspects of glucose isomerase.

Publisher Summary Metal ions serve a variety of functions in proteins. The most important function is to enhance the structural stability of the protein in the conformation required for biological function and to take part in the catalytic processes of enzymes. Metal ions can take part in trigger and control mechanisms by specifically altering or stabilizing a macromolecular conformation on binding. This chapter discusses the properties of metals that are useful in the structure and function of proteins, particularly enzymes, and the geometry of interaction of metals with the various chemical groups of proteins are emphasized. Ligands donate an electron pair to the bond and are generally negatively charged or neutral. Important in a study of metal-ligand interactions are the polarizability of both the metal ion and the ligand, the number of the ligands around each metal ion, and the stereochemistry of the resulting arrangement. The stereochemistry of liganding of metal ions in proteins is known for several proteins. Some selected examples follow with data derived from the Protein Data Bank are emphasized in the chapter. The metals in protein crystal structures discussed are— namely, (1) copper, (2) iron, (3) manganese, (4) zinc, (5) magnesium, and (6) calcium. In the cases in which two different metals are bound, information can be obtained on preferential sites for each metal in the presence of the other.

Structural Aspects of Metal Liganding to Functional Groups in Proteins

Most enzymatic reactions have very large and remarkably similar apparent second-order rate constants, kcat/KM, at mean values of about 107 M−1 s−1 with kcat in the range of 10–1000 s−1.1–3 In fact, many reactions approach the diffusional encounter rate at the limited enzyme concentration (<10−5 M) in the cell.4 Wolfenden illustrated the catalytic power of enzymes by comparing the rate constant of the catalyzed reaction with that of the same reaction in the absence of the enzyme in aqueous solution, kaq.2,5 Evidently, the most proficient enzymes are those catalyzing the slowest spontaneous reactions, such as the hydrolysis of glycosides and phosphate esters and the decarboxylation reactions of amino acids and of orotidine 5′-monophosphate (OMP), as catalyzed by OMP decarboxylase (ODC).2 In the latter case, the unimolecular rate constant of the spontaneous decarboxylation of OMP is accelerated (kcat/kaq) by 17 orders of magnitude in the active site of ODC.5,6 This reaction also has the distinction of being among the most proficient enzymes in catalyzing reactions without the involvement of cofactors. Significantly, Wolfenden’s experimental approach has been followed and paralleled in computational studies.7 The experiments, along with computational results that we review in this article, provide abundant evidence that the very large observed reductions of the free energy of activation can be achieved through the strong synergism of enzyme and substrate interactions “using ordinary noncovalent forces of attraction”,8 although in other cases enzyme catalysis may involve covalent intermediates9 or a change in reaction mechanism as compared to aqueous solution.

Noncovalent attractive forces are mainly electrostatic in nature; they include ion pair interactions, hydrogen bonding, and electronic polarization. The competition between solvent–solvent and solvent–solute interactions contributes to hydrophobic effects (where the “solute” is the substrate or any part of the protein or a coenzyme that participates in the reaction coordinate and the “solvent” is water, spectator residues of the enzyme in the active site, and faraway parts of the protein or protein complex). These interactions all contribute to catalysis. It has been argued insightfully that electrostatic preorganization effects are a key source of enzyme catalysis,10 but the questions remain of what other factors contribute and how preorganization is arranged such that the transition state is stabilized preferentially to the reactant state. To understand enzyme catalysis and mechanism, it is necessary, and often challenging, to elucidate the unique ways in which each enzyme exerts electrostatic and other forces on the substrate and the transition state.

In the past 10 years, many computational studies of enzymatic reactions have been carried out, combining quantum mechanical, classical mechanical, and statistical mechanical techniques, coupled with advances in protein structure determination, site-directed mutagenesis, and fast computers and algorithms. All computational studies of atomic scale dynamics must begin with a potential energy surface, and the most promising approach to calculating this surface is to treat the enzyme active site by electronic structure methods11–19 that include the electronic polarization of the reactive species by the dynamical fluctuations of the enzyme–solvent environment through effective sampling of the enzyme conformational space. Although a review necessarily involves only a very limited selection of the reactions that enzymes catalyze in the cell, we can nevertheless conclude7 that each enzyme has its unique characteristics, and enzymes use all possible means to achieve the ultimate objective of reducing the free energy of activation.

In addition to providing an enormous rate acceleration, enzymes exercise precise control over the regio- and stereochemistry of the reactions that they catalyze, an aspect of enzyme catalysis that has received relatively little attention in computations (recent studies of triosephosphate isomerase and glyoxal synthase provide a noteworthy exception20). This control is perhaps best illustrated by the reactions catalyzed by terpenoid synthases,21 a large group of enzymes that transform a limited number of linear substrates such as geranyl diphosphate (C10), farnesyl diphosphate (C15), and geranylgeranyl diphosphate (C20) to tens of thousands natural products with a variety of rings and stereocenters, presumably by prefolding the same substrate to a “proper” conformation in the unique binding pocket of each enzyme and subsequently preventing the highly reactive carbocation intermediates from undergoing side reactions and preventing premature terminations of the catalyzed reaction sequences. Both experimental and computational studies appear to point to an important role for the balance of thermodynamic and kinetic factors along the cyclization cascade.22,23 Thus, it is of great interest not only to understand the origin of the enormous catalytic power of enzymes that they achieve by lowering the free energy of activation but also to characterize the detailed mechanism of enzyme actions that control each reaction step and provide the desired regio- and stereospecificity.

In this review, we summarize computational studies of the mechanisms and free energies of selected enzymatic reactions. We first highlight computational approaches for enzymatic reactions, with special emphasis on two key elements that affect the computational accuracy, namely, the potential energy function and statistical mechanical sampling of the enzyme system. The potential energy functions may be based on quantum mechanical models, or they may be based on molecular mechanics force fields. In either case, to achieve the required accuracy to understand catalysis, it is essential to parametrize and validate the potential energy functions (or, equivalently, the methods used to calculate them) against model reactions and specific hydrogen bonding interactions in the gas phase. Only when the performance of the potential functions on the intrinsic reactivity of the chemical reactions has been justified can one begin to address the key questions of solvent effects and enzyme catalysis through molecular dynamics and free energy simulations. We then discuss a third element, namely, the choice of the reaction coordinate for determining the free energy of activation to characterize the mechanism of enzymatic processes. Then, we illustrate a variety of factors that have been found to contribute to catalysis in specific enzymatic reactions by lowering the free energy of activation relative to that for the uncatalyzed process in aqueous solution. Finally, we provide a summary of the major conclusions.

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Mechanisms and free energies of enzymatic reactions.

The structures of crystalline D-xylose isomerase (D-xylose ketol-isomerase; EC 5.3.1.5) from Streptomyces rubiginosus and of its complexes with substrate and with an active-site-directed inhibitor have been determined by x-ray diffraction techniques and refined to 1.9-A resolution. This study identifies the active site, as well as two metal-binding sites. The metal ions are important in maintaining the structure of the active-site region and one of them binds C3-O and C5-O of the substrate forming a six-membered ring. This study has revealed a very close contact between histidine and C1 of a substrate, suggesting that this is the active-site base that abstracts a proton from substrate. The mechanism-based inhibitor is a substrate analog and is turned over by the enzyme to give a product that alkylates this same histidine, reinforcing our interpretation. The changes in structure of the native enzyme, the enzyme with bound substrate, and the alkylated enzyme indicate that the mechanism involves an "open-chain" conformation of substrate and that the intermediate in the isomerization reaction is probably a cis-ene diol because the active-site histidine is correctly placed to abstract a proton from C1 or C2 of the substrate. A water molecule binds to C1O and C2O of the substrate and so may act as a proton donor or acceptor in the enolization of a ring-opened substrate.

https://www.pnas.org/content/pnas/86/12/4440.full.pdf

Vincent Burger

Papers

X-ray analysis of D-xylose isomerase at 1.9 A: native enzyme in complex with substrate and with a mechanism-designed inactivator