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PubMed1999

Catalysis by metal-activated hydroxide in zinc and manganese metalloenzymes.

The metal-activated hydroxide ion is a critical nucleophile in metalloenzymes that catalyze hydrolysis or hydration reactions. The most common metal used is zinc; occasionally, other transition metals such as manganese are required. Human carbonic anhydrase II and rat liver arginase serve as well-studied paradigms of zinc and manganese metalloenzymes, respectively. Comparative structure-function relationships between these two metalloenzymes highlight parallels in the chemistry of metal-activated hydroxide: (a) the protein environment of metal-bound hydroxide modulates its reactivity; (b) a hydrogen bond with metal-bound hydroxide holds it in the proper orientation for catalysis; (c) nonmetal substrate-binding sites are implicated in both enzyme mechanisms; and (d) regeneration of metal-bound hydroxide ion from a metal-bound water molecule requires proton transfer to bulk solvent mediated by a histidine proton shuttle residue. Interestingly, the electrostatics of catalysis differ between the two enzymes, in that the first step of catalysis requires formation of a negatively charged transition state in the carbonic anhydrase II mechanism, whereas a neutral transition state is approached in the arginase mechanism. This electrostatic feature may contribute to the differences in the chemistry, the metal binding sites, and the metal specificity between these two enzymes.

Animals↗
PubMed2006

Testing electrostatic complementarity in enzyme catalysis: hydrogen bonding in the ketosteroid isomerase oxyanion hole.

A longstanding proposal in enzymology is that enzymes are electrostatically and geometrically complementary to the transition states of the reactions they catalyze and that this complementarity contributes to catalysis. Experimental evaluation of this contribution, however, has been difficult. We have systematically dissected the potential contribution to catalysis from electrostatic complementarity in ketosteroid isomerase. Phenolates, analogs of the transition state and reaction intermediate, bind and accept two hydrogen bonds in an active site oxyanion hole. The binding of substituted phenolates of constant molecular shape but increasing pK(a) models the charge accumulation in the oxyanion hole during the enzymatic reaction. As charge localization increases, the NMR chemical shifts of protons involved in oxyanion hole hydrogen bonds increase by 0.50-0.76 ppm/pK(a) unit, suggesting a bond shortening of 0.02 A/pK(a) unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates (DeltaDeltaG = -0.2 kcal/mol/pK(a) unit). The small effect of increased charge localization on affinity occurs despite the shortening of the hydrogen bonds and a large favorable change in binding enthalpy (DeltaDeltaH = -2.0 kcal/mol/pK(a) unit). This shallow dependence of binding affinity suggests that electrostatic complementarity in the oxyanion hole makes at most a modest contribution to catalysis of 300-fold. We propose that geometrical complementarity between the oxyanion hole hydrogen-bond donors and the transition state oxyanion provides a significant catalytic contribution, and suggest that KSI, like other enzymes, achieves its catalytic prowess through a combination of modest contributions from several mechanisms rather than from a single dominant contribution.

Anions↗
PubMedDate not supplied

Catalytic antibodies for complex reactions: hapten design and the importance of screening for catalysis in the generation of catalytic antibodies for the NDA/CN reaction.

Success in generating catalytic antibodies as enzyme mimics lies in the strategic design of the transition-state analog (TSA) for the reaction of interest, and careful development of screening processes for the selection of antibodies that are catalysts. Typically, the choice of TSA structure is straightforward, and the criterion for selection in screening is often binding of the TSA to the antibody in a microtiter-plate assay. This article emphasizes the problems of TSA design in complex reactions and the importance of selecting antibodies on the basis of catalysis as well as binding to the TSA. The target reaction is the derivatization of primary amines with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide ion. The desired outcome is selective catalysis of formation of the fluorescent derivative in preference to nonfluorescent side-products. In the study, TSA design was directed toward the reaction branch leading to the fluorescent product. Here, we describe a microtiter plate-based assay that is capable of detecting antibodies showing catalytic activity at an early stage. Of the antibodies selected, 36% showed no appreciable binding to any of the substrates tested, but did show catalytic activity in derivatizing one or more of the amino acids screened. In contrast, only two out of 77 clones that showed binding did not show catalysis. Thus, in this complex system, observation of binding is a good predictor of the presence of catalytic activity, and failure to observe binding is a poor predictor of the absence of catalytic activity.

Animals↗
PubMedDate not supplied

The mechanism of DNA cleavage by the type II restriction enzyme EcoRV: Asp36 is not directly involved in DNA cleavage but serves to couple indirect readout to catalysis.

Three different mechanisms have been proposed to describe DNA cleavage by the type II restriction endonuclease EcoRV, which differ in the number and function of metal ions directly involved in catalysis and the different roles assigned to amino acid residues in the active sites and a phosphate group of the substrate. There are only four acidic amino acid residues close to the scissile bond: the essential Asp74 and Asp90, the non-essential Glu45, and Asp36. We show here that Asp36 can be exchanged for alanine, with only minor effects on the cleavage rate of the nearby phosphodiester bond, excluding that Asp36 could be directly involved in catalysis. Hence, the two versions of the two-metal-ion mechanism are not compatible with the experimental data, because too few ligands for two metal ions are present near the active site of EcoRV. Our result, thus, supports the one-metal-ion mechanism for EcoRV. We suggest that Asp36 has an allosteric effect by which specific contacts between one strand of the DNA and one subunit of the enzyme trigger the activation of one catalytic center. Given the similar structures of the active sites of EcoRV, EcoRI, BamHI, PvuII and FokI, as well as the occurrence of a characteristic catalytic motif in several other restriction enzymes, we conclude that these enzymes most likely share a similar mechanism of DNA cleavage, whose characteristic feature is the involvement of only one Mg2+ ion in catalysis.

Aspartic Acid↗
PubMed1996

Antibody catalysis via strategic use of haptenic charge.

General acid-base catalysis contributes substantially to the efficacy of many enzymes. Similar effects can be exploited in antibody catalysis by taking advantage of charge complementarity between immunoglobulin and hapten (the template used to induce the antibody) to elicit functional groups in the combining site. This strategy has proved useful in the catalysis of a diverse set of chemical transformations, including elimination reactions. Provided that hapten design is optimized and the immune response is screened extensively, the efficiency of the resulting antibody catalysts can rival that of analogous natural enzymes.

Alkenes↗
PubMed1999

The origins of heterogeneous catalysis by platinum: Johann Wolfgang Döbereiner's contributions.

A number of examples of what Jöns Jacob Berzelius was later (1835) to call catalysis, have been known from antiquity. One of the most prominent of these, dating from the early eighteenth century, was the action of platinum black on hydrogen observed in 1823 by Johann Wolfgang Döbereiner (1780-1849), Professor of Chemistry and Technology at the Universität Jena (1810-1849), who is regarded as the discoverer of platinum catalysis. His startling discovery led to his invention of the widely used pneumatic gas lighter (Döbereinersches Feuerzeug) and was cited by Berzelius in his formulation of the concept of catalysis.

Catalysis↗
PubMed2001

Ribozymes of the hepatitis delta virus: recent findings on their structure, mechanism of catalysis and possible applications.

Although the delta ribozymes have been studied for more than ten years the most important information concerning their structure and mechanism of catalysis were only obtained very recently. The crystal structure of the genomic delta ribozyme turns out to be an excellent example of the extraordinary properties of RNA molecules to fold into uniquely compact structures. Details of the X-ray structure have greatly stimulated further studies on the folding of the ribozymes into functionally active molecules as well as on the mechanism of RNA catalysis. The ability of the delta ribozymes to carry out general acid-base catalysis by nucleotide side chains has been assumed in two proposed mechanisms of self-cleavage. Recently, considerable progress has been also made in characterizing the catalytic properties of trans-acting ribozyme variants that are potentially attractive tools in the strategy of directed RNA degradation.

Base Sequence↗
PubMed2001

Polymer supports in organic catalysis and synthesis.

Recent developments in the preparation of polymer supports for synthesis and catalysis are reviewed. The use of insoluble supports for synthesis of one molecule per bead and chemistry leading to new ways to use insoluble supports are discussed. New routes to design soluble, high capacity supports for synthesis are highlighted and the roles of polymers in catalysis, both for re-use and separation, are discussed using metathesis catalysts and Pd(0) catalysts as examples. The role of dendrimers as supports for synthesis and catalysis are reviewed, and new separation schemes using inverse temperature-dependent solubility of polymers and liquid/liquid separations are discussed.

Catalysis↗
PubMedDate not supplied

[Dynamic theory of enzymatic catalysis].

Formal kinetics of an enzymatic reaction is considered in terms of a dynamic scheme of catalysis. Conformation reconstructions of a liberated enzyme and e-s-complex are analysed upon the phase plane. An expression for the enzyme stationary activity depending on the rate of conformation reconstructions is obtained. The case of purely dynamic catalysis is analysed and possible initiation of enzymatic activity variation in time connected with initial synchronization of molecules is shown. This phenomenon is considered as a possible criterion of the dynamic character of enzymatic catalysis.

Catalysis↗
PubMed1991

Free energy perturbation calculations on binding and catalysis after mutating threonine 220 in subtilisin.

We present the results of free energy perturbation calculations on binding and catalysis of a tetrapeptide substrate, acetyl-Phe-Ala-Ala-Phe-NMe, by native subtilisin BPN' and a subtilisin BPN' mutant (Thr220----Ala220). The calculated difference in the free energy of binding was 0.70 +/- 0.72 kcal/mol. The calculated difference in the free energy of catalysis was 1.48 +/- 0.89 kcal/mol. These calculated values compare well with the experimental values in which another substrate, succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, was used. These findings suggest that Thr220 is more important for catalysis than substrate binding.

Amino Acid Sequence↗
PubMed1987

Studies of the mechanism of glutamine synthetase utilizing pH-dependent behavior in catalysis and binding.

The pKa values of enzyme groups of Escherichia coli glutamine synthetase which affect catalysis and/or substrate binding were determined by measuring the pH dependence of Vmax and V/K. Analysis of these data revealed that two enzyme groups are required for catalysis with apparent pKa values of approximately 7.1 and 8.2. The binding of ATP is essentially independent of pH in the range studied while the substrate ammonia must be deprotonated for the catalytic reaction. Using methylamine and hydroxylamine in place of ammonia, the pKa value of the deprotonated amine substrate as expressed in the V/K profiles was shifted to a lower pKa value for hydroxylamine and a higher pKa value for methylamine. These data indicate that the amine substrate must be deprotonated for binding. Hydroxylamine is at least as good a substrate as ammonia judged by the kinetic parameters whereas methylamine is a poor substrate as expressed in both the V and V/K values. Glutamate binding was determined by monitoring fluorescence changes of the enzyme and the data indicate that a protonated residue (pKa = 8.3 +/- 0.2) is required for glutamate binding. Chemical modification by reductive methylation with HCHO indicated that the group involved in glutamate binding most likely is a lysine residue. In addition, the Ki value for the transition state analog, L-3-amino-3-carboxy-propanesulfonamide was measured as a function of pH and the results indicate that an enzyme residue must be protonated (pKa = 8.2 +/- 0.1) to assist in binding. A mechanism for the reaction catalyzed by glutamine synthetase is proposed from the kinetic data acquired herein. A salt bridge is formed between the gamma-phosphate group of ATP and an enzyme group prior to attack by the gamma-carboxyl of glutamate on ATP to form gamma-glutamyl phosphate. The amine substrate subsequently attacks gamma-glutamyl phosphate resulting in formation of the tetrahedral adduct before phosphate release. A base on the enzyme assists in the deprotonation of ammonia during its attack on gamma-glutamyl phosphate or after the protonated carbinol amine is formed. Based on the kinetic data with the three amine substrates, catalysis is not rate-limiting through the pH range 6-9.

Adenosine Triphosphate↗
PubMed1987

Catalysis of thrombin inhibition provides an index for estimating the antithrombotic potential of glycosaminoglycans in rabbits.

Previous studies have demonstrated that standard anticoagulant tests are poor indices of the antithrombotic potential of glycosaminoglycans which are weak catalysts of the thrombin-antithrombin III reaction. In this study we investigated whether the catalysis of thrombin inhibition by plasma could serve as a reliable index for assessing the antithrombotic effectiveness of glycosaminoglycans. Equal volumes of 125I-thrombin and control or test plasma were incubated for up to 10 min at 37 degrees C. Inactivation of thrombin was then determined after 7.9% SDS-polyacrylamide gel electrophoresis and subsequent autoradiography. Increasing concentrations of heparin (greater than 0.066 micrograms/mL or 0.01 USP units/mL) and dermatan sulfate (greater than 0.1 micrograms/mL) could be readily demonstrated in undiluted plasma by enhanced formation of complexes of thrombin with antithrombin III and heparin cofactor II respectively. However, the detection of any catalytic effect of the two glycosaminoglycans decreased significantly with increasing plasma dilutions. When ex vivo plasmas obtained from rabbits that had been injected with the minimum dose of any one of seven glycosaminoglycans required to achieve their optimal antithrombotic effect were assessed for their ability to catalyse thrombin inhibition, there was approximately a 2-fold increase in the amount of thrombin inactivated 30 s after the thrombin had been added to the plasma. The enhanced inhibition of thrombin was achieved by catalysis of antithrombin III and/or heparin cofactor II activities. These results suggest that measurement of the catalysis of thrombin inactivation in undiluted plasma is a sensitive and reliable index for estimating the antithrombotic potential of glycosaminoglycans in rabbits.

Animals↗
PubMed1987

Action mechanism of Escherichia coli DNA photolyase. II. Role of the chromophores in catalysis.

DNA photolyase repairs pyrimidine dimers in DNA in a reaction that requires visible light. Photolyase from Escherichia coli is normally isolated as a blue protein and contains 2 chromophores: a blue FAD radical plus a second chromophore that exhibits an absorption maximum at 360 nm when free in solution. Oxidation of the FAD radical is accompanied by a reversible loss of activity which is proportional to the fraction of the enzyme flavin converted to FADox. Quantitative reduction of the radical to fully reduced FAD causes a 3-fold increase in activity. The results show that a reduced flavin is required for activity and suggest that flavin may act as an electron donor in catalysis. Comparison of the absorption spectrum calculated for the protein-bound second chromophore (lambda max = 390 nm) with fluorescence data and with the relative action spectrum for dimer repair indicates that the second chromophore is the fluorophore in photolyase and that it does act as a sensitizer in catalysis. On the other hand, enzyme preparations containing diminished amounts of the second chromophore do not exhibit correspondingly lower activity. This suggests that reduced flavin may also act as a sensitizer in catalysis. The blue color of the enzyme is lost upon reduction of the FAD radical. The fully reduced E. coli enzyme exhibits absorption and fluorescence properties very similar to yeast photolyase. This indicates that the two enzymes probably contain similar chromophores but are isolated in different forms with respect to the redox state of the flavin.

Catalysis↗
PubMed1994

Catalysis by Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Proposed roles of histidine 865, glutamate 558, and aspartate 766.

We employed the overexpressed catalytic domains of wild-type Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) and of mutant enzymes E558Q, H865Q, and D766N to investigate the roles of Glu558, His865, and Asp786 in catalysis. Five reactions were studied: reductive deacylation of HMG-CoA or of mevaldyl-CoA to mevalonate, reduction of mevaldehyde to mevalonate, oxidation of mevaldyl-CoA to HMG-CoA, and oxidative acylation of mevaldehyde to HMG-CoA. While only the wild-type enzyme catalyzed all five reactions, mutant enzymes E558Q and H865Q catalyzed other reactions at significant rates. Mutant enzyme D766N, although apparently structurally similar to the wild-type enzyme, was inactive for all five reactions. While an ineffective catalyst for the overall reaction, mutant enzyme H865Q catalyzed the reduction of mevaldehyde to mevalonate at about the wild-type rate. Coenzyme A, which stimulated mevaldehyde reduction by the wild-type enzyme, inhibited mutant enzyme H865Q, apparently due to an impaired ability to protonate the coenzyme A thioanion, CoAS-. Based on these data, we infer that Glu558, His865, and Asp766 function in catalysis at the indicated stage of the overall reaction, [formula: see text] and we propose a revised mechanism for catalysis by mammalian HMG-CoA reductases.

Amino Acid Sequence↗
PubMed1998

The role of active site flexibility in enzyme catalysis.

It has been shown in this and other laboratories that during the unfolding of a number of enzymes inactivation generally precedes global unfolding of the enzyme molecule, leading to the suggestion that enzyme active sites are usually more "fragile" and more easily "perturbed" than the molecule as a whole and are therefore conformationally more flexible than the rest of the molecule. However, the role of active site flexibility in enzyme catalysis still remains to be explored. In the induced fit hypothesis originally proposed by Koshland, the presence of the substrate induces a conformational change at the active site so as to fit with the structure of the substrate. By X-ray crystallographic structural analysis of E. coli dihydrofolate reductase liganded with cofactors and substrates, Sawaya and Kraut showed the enzyme in different conformational states indeed while complexed with different ligands, suggesting that the enzyme molecule passes through different conformational states through the whole process of catalysis. Muscle lactate dehydrogenase can be stabilized either in concentrated ammonium sulfate or by cross-linking with glutaraldehyde together with a decrease in enzyme activity which can be restored to the original level in dilute guanidine hydrochloride possibly by increased flexibility at the active site. It is known that a number of enzymes can be activated by chaotropic agents such as urea or guanidine hydrochloride. The activation of dihydrofolate reductase by either urea or guanidine hydrochloride is accompanied by an increase in susceptibility to proteolysis. Isolation of the tryptic peptides of the activated enzyme and sequence analysis allowed identification of the sites of proteolysis to be at or near the active site of the enzyme, indicating an opening up of the active site conformation in the activated state. All the above indicate that active site flexibility plays an important role in enzyme catalysis. It is possible that during the catalytic cycle, the enzyme molecule passes through different stages and each stage requires the molecule to be in a different conformation, especially at the active site. Rapid transition between the different conformational states, and hence the flexibility of the active site, is therefore mandatory for the maximal expression of enzyme activity.

Animals↗
PubMed1998

Mutational analysis of F1F0 ATPase: catalysis and energy coupling.

Escherichia coli ATP synthase has eight subunits and functions through transmission of conformational changes between subunits. Extensive mutational analyses identified essential residues for catalysis and conformation transmission. Pseudorevertant studies revealed that beta/alpha and beta/gamma subunits interactions are important for the energy coupling between catalysis and H+ translocation. In this article, we discuss mechanism of catalysis and energy coupling based on our recent mutation studies.

Amino Acid Sequence↗
PubMed2002

"Asymmetric" catalysis by lanthanide complexes.

Catalysis with lanthanide (Ln) complexes has been underestimated for long time, although Ln(III) complexes have great advantages as Lewis acid catalysts for "asymmetric" carbon-carbon bond-forming reactions. Lanthanide complexes are highly active in ligand-substitution reactions, especially with hard ligands. The association with substrates and dissociation of products are achieved fast enough for high catalyst efficiency. The asymmetric catalysis of organic reactions can be greatly advanced by the use of Ln complexes with chiral ligands such as binaphthol (binol). Ln(II) complexes are good reducing agents, which can be used in a wide variety of synthetically important reactions; when chiral ligands are used, many of these reactions are highly stereoselective. In the context of "green chemistry", the development of asymmetric Ln catalysts, and their recyclable use, is of increasing importance. This review gives an overview of the most recent developments in catalysis with lanthanide(II) and lanthanide(III) complexes.

Journal Article↗
PubMed2003

Recycling of homogeneous hydrogenation catalysts by dialysis coupled catalysis.

Although transition-metal complexes are very attractive as homogeneous catalysts in fine chemistry, their high prices often limit their applications. A means to recycle those catalysts would solve this problem and would simultaneously facilitate the downstream purification of the product. This is now realized in a new concept in which homogeneous catalysis is coupled to dialysis. The advantages of homogeneous catalysis (off-the-shelf catalysts, high activities and selectivities) are thus combined with those of heterogeneous catalysis (easy catalyst separation from product solution, reuse of catalyst, and possibility for continuous operation). Since the heart of the process is the membrane, self-prepared membranes were preferred as they allow a better control and understanding of the separation characteristics. Rhodamine B was used as a probe molecule to define the working conditions of the membrane. The concept is proven to work for two relevant chiral reactions: a hydrogenation with Ru-BINAP and a hydrogen transfer reaction with Ru-TsDPEN [BINAP=(1,1'-binaphthalene)-2,2'-diylbis(diphenylphosphine); TsDPEN= tosyl-N,N'-diphenyl-1,2-ethanediamine].

Journal Article↗