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At least 271 records · Page 15Linked to original sources
PubMed2006

The involvement of Arg265 of mouse ribonucleotide reductase R2 protein in proton transfer and catalysis.

Ribonucleotide reductase class I enzymes consist of two non-identical subunits, R1 and R2, the latter containing a diiron carboxylate center and a stable tyrosyl radical (Tyr*), both essential for catalysis. Catalysis is known to involve highly conserved amino acid residues covering a range of approximately 35 A and a concerted mechanism involving long range electron transfer, probably coupled to proton transfer. A number of residues involved in electron transfer in both the R1 and R2 proteins have been identified, but no direct model has been presented regarding the proton transfer side of the process. Arg265 is conserved in all known sequences of class Ia R2. In this study we have used site-directed mutagenesis to gain insight into the role of this residue, which lies close to the catalytically essential Asp266 and Trp103. Mutants to Arg265 included replacement by Ala, Glu, Gln, and Tyr. All mutants of Arg265 were found to have no or low catalytic activity with the exception of Arg265 to Glu, which shows approximately 40% of the activity of native R2. We also found that the Arg mutants were capable of stable tyrosyl radical generation, with similar kinetics of radical formation and R1 binding as native R2. Our results, supported by molecular modeling, strongly suggest that Arg265 is involved in the proton-coupled electron transfer pathway and may act as a proton mediator during catalysis.

Animals↗
PubMed1987

Single site catalysis of the F1-ATPase from Saccharomyces cerevisiae and the effect of inorganic phosphate on it.

The kinetical characteristics of ATP hydrolysis by mitochondrial F1-ATPase from Saccharomyces cerevisiae (yeast) have been studied under conditions where only a single catalytic site per enzyme molecule bound ATP. Four major features were observed, that is, fast ATP binding to the enzyme, slow product release from the enzyme, an equilibrium close to unity between ATP and products on the enzyme, and promotion of ATP hydrolysis on the second addition of a large excess of ATP (cold chase). These are essentially the same as the kinetical characteristics observed for beef heart mitochondrial F1-ATPase, which were called as unisite catalysis by Grubmeyer et al. (Grubmeyer, C. et al. (1982) J. Biol. Chem. 257, 12092-12100), although the release of a hydrolysis product, Pi, from the yeast enzyme appeared to occur significantly faster than that from the beef enzyme, which resulted in a decreased extent of cold chase promotion of ATP hydrolysis of the yeast enzyme. The yeast F1-ATPase showed unisite catalysis even in the absence of Pi in the reaction mixtures, while it was reported for the beef F1-ATPase that the presence of Pi in the reaction mixture was essential for unisite catalysis (Penefsky, H.S. & Grubmeyer, C. (1984) in H+-ATPase (ATP Synthase) (Papa, S. et al., eds.) pp. 195-204, The ICSU Press). Another difference in the Pi effect on the kinetics was that ATP hydrolysis was initiated without a lag time in the absence of Pi in the case of the yeast enzyme when a 1,000-fold molar excess of ATP per enzyme molecular was mixed with the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1992

Escherichia coli F0F1-ATPase. Residues involved in catalysis and coupling.

The molecular biological approach has provided important information toward understanding the complexities of the F0F1 ATPase. This article focuses on our recent results on the ATPase catalytic site contained in the beta subunit and the role of the gamma subunit in regulation of proton transport. We used a combination of affinity labeling and mutagenesis to locate several residues of the alpha and beta subunits in the catalytic site. Adenosine triphosphopyridoxal (AP3-PL) labeled beta Lys-155, beta Lys-201 and alpha Lys-201, suggesting that they are near the gamma-phosphate moiety of ATP. Turning to a mutagenesis approach we demonstrated that the two conserved residues, beta Lys-155 and beta Thr-156 in the glycine-rich sequence, are essential for catalysis. Finally, using pseudorevertant analysis, we positioned residue beta Gly-149 (also in the glycine-rich sequence) in proximity to beta Ser-174, beta Glu-192 (binding site for DCCD), and beta Val-198 (only three residues away from the AP3-PL binding site, beta Lys-201). Genetic studies suggested that the gamma subunit plays a role in regulation of catalysis and its coupling with proton conduction. We found that four mutations in the carboxyl-terminal region (gamma Gln-269-->Leu, gamma Gly-275-->Lys, gamma Thr-277-->end, or frameshift) had similar membrane ATPase activities but different ATP-dependent proton pumping and growth by oxidative phosphorylation. These results suggested a perturbation in the coupling between catalysis and proton translocation. We were able to clearly define the "uncoupling" by introducing mutations in the amino-terminal region of the gamma subunit. We were led to gamma Met-23-->Lys and Arg which resulted in an enzyme still regulated by delta microH+, but with profoundly inefficient coupling between ATPase catalytic sites and proton translocation in both ATP-dependent proton pumping and delta microH(+)-driven ATP synthesis. Second-site mutations in the carboxyl-terminal region of the gamma subunit reversed this effect.

Amino Acid Sequence↗
PubMed1980

Heterogeneous catalysis by phospholipase A2: mechanism of hydrolysis of gel phase phosphatidylcholine.

Hydrolysis of gel phase dipalmitoylphosphatidylcholine (DPPC) at 37 degrees C catalysed by Crotalus atrox phospholipase A2 (PLA) is described extremely well by the "path 1" kinetic mechanism of Tinker and Wei (1979) (Can. J. Biochem. 57, 97-106), if reversible adsorption is allowed as a side reaction. Progress curves show an initial rapid phase, the initial velocity being a Michaelis-Menten function dependent on the catalytic properties of the enzyme (kcat approximately equal to 9200 min-1, Km approximately equal to 0.12 mM), then level off to a slower rate determined by the desorption equilibrium constant (KD approximately equal to 0.01 mM) and desorption rate constant (kD approximately equal to 0.15 min-1). The relaxation time, tau, for the transition to the desorption-limited reaction is approximately 0.5 min; this large value of tau probably arises from a slow conversion of active, dimeric enzyme to an inactive protein species adsorbed to the lipid surface. At later times in the reaction there is an increase in the rate of hydrolysis, attributed to a stimulation of desorption by the products. The desorption equilibrium constant KD is a quadratic function of the surface concentration of products and increases 20- to 30-fold when all accessible substrate is hydrolysed. Both lysophosphatidylcholine (lyso-PC) and fatty acid were found to stimulate the desorption, but lyso-PC was also found to be a competitive inhibitor of the catalysis. Adsorption of PLA to DPPC and egg PC vesicles was directly measured using a gel partition technique. Strong binding to egg PC was observed, which was not dependent on the presence of calcium ion (essential for catalysis); PLA inhibited by acylation of up to four lysine residues per mole of monomeric enzyme with ethoxyformic anhydride was equally strongly adsorbed, indicating that lipid binding is not dependent on catalytic activity. Reaction products greatly weakened the binding of PLA to the lipid surface as expected. Cholesterol had two effects on the hydrolytic reaction: there was a striking decrease in the rate of the slower, desorption-limited phase, the rate of which decrease to almost zero at 15 mol% cholesterol, but there was also evidence for the formation of a complex with stoichiometry 1 cholesterol: 2 DPPC in which DPPC is no longer a substrate for the enzyme. Implications of the proposed mechanism for specificity and control of surface catalysis by PLA are discussed.

Crotalid Venoms↗
PubMed2001

Combinatorial Heterogeneous Catalysis-A New Path in an Old Field.

Combinatorial catalysis is the systematic preparation, processing, and testing of large diversities of chemically and physically different materials libraries in a high-throughput fashion. It also embodies microfabrication, robotics, automation, instrumentation, computational chemistry, and large-scale information management (informatics), and as such carries the promise of a renaissance in catalytic reaction engineering. Significant progress has already been made in demonstrating the speed and economic advantage of combinatorial approaches by the discovery of superior catalytic materials in a matter of hours and days, as opposed to the months and years required using traditional methods. Combinatorial methods can also significantly contribute to our understanding of catalytic function by increasing our chances of discovering totally new and unexpected catalytic materials, and by expediting the recognition of trends and patterns of structure-activity relations, from which new catalytic materials can be designed more efficiently. Combinatorial catalysis undoubtedly will be the new paradigm of catalysis research as the industry faces increasing global competition and pressure for the development of environmentally friendly processes at a time when resources for research are diminishing.

Journal Article↗
PubMed1987

The defective proton-ATPase of uncA mutants of Escherichia coli. Identification by DNA sequencing of residues in the alpha-subunit which are essential for catalysis or normal assembly.

A group of mutant uncA alleles, affecting essential residues of the alpha-subunit of Escherichia coli proton-ATPase, have been identified by intragenic complementation mapping, cloning, and DNA sequencing. One of the mutations, uncA450, abolishes normal assembly of F1-ATPase. The amino acid substitution found was Glu-299----Lys, which is predicted to lie in an alpha-helix in alpha-subunit. The reversal of the charge at residue 299 is a likely cause of defective assembly. The uncA462 allele causes impairment of catalysis while allowing normal assembly of membrane-bound F1-ATPase. The amino acid substitution found was Ser-347----Phe. Three mutations which impair catalysis but do not cause structural perturbation of either membrane-bound or solubilized F1ATPase were characterized as follows: uncA401, Ser-373----Phe; uncA447, Gly-351----Asp; uncA453, Ser-375----Phe. We predict here that the nucleotide-binding domain of alpha-subunit is formed by the amino acids in the sequence from residue 160 to approximately residue 340. The mutations which cause impairment of catalysis lie in a short segment between residues 347-375 of alpha-subunit, at the C-terminal end of the predicted nucleotide-binding domain. This segment is suggested to be important for beta-alpha-beta intersubunit conformational interaction involved in positive catalytic cooperativity in F1-ATPase.

Alleles↗
PubMed1982

Conversion of 4-hydroperoxycyclophosphamide and 4-hydroxycyclophosphamide to phosphoramide mustard and acrolein mediated by bifunctional catalysis.

The rates at which 4-hydroperoxycyclophosphamide and 4-hydroxycyclophosphamide are converted to phosphoramide mustard and acrolein were determined as a function of buffer composition, buffer concentration, and pH. Conversion of 4-hydroperoxycyclophosphamide to 4-hydroxycyclophosphamide in 0.5 M Tris buffer, pH 7.4, 37 degrees, was first-order (k = 0.016 min-1), but subsequent conversion of 4-hydroxycyclophosphamide to phosphoramide mustard and acrolein under these conditions was negligible. Phosphoramide mustard and acrolein were readily generated from 4-hydroperoxycyclophosphamide or 4-hydroxycyclophosphamide when either of these agents was placed in phosphate buffer. Conversion of 4-hydroxycyclophosphamide to phosphoramide mustard and acrolein was first-order with respect to 4-hydroxycyclophosphamide (k = 0.126 min-1 in 0.5 M phosphate buffer, pH 8, 37 degrees) as well as first-order with respect to phosphate serving as a catalyst. The rate-determining step in the reaction was pH dependent only insofar as the hydrogen ion concentration governed the relative amounts of monobasic and dibasic phosphate present. Pseudo-first-order rate constants were 0.045 M-1 min-1 for monobasic phosphate and 0.256 M-1 min-1 for dibasic phosphate. The role of phosphate in this reaction was as that of a bifunctional catalyst. The reaction was not subject to specific or general, acid or base, catalysis. Other bifunctional catalysts such as glucose-6-phosphate and bicarbonate also catalyzed the reaction, albeit less efficiently. Aldophosphamide apparently exists only transiently; its presence could not be established by 31P nuclear magnetic resonance spectroscopy. We conclude that, in the reaction sequence 4-hydroxycyclophosphamide leads to aldophosphamide leads to phosphoramide mustard + acrolein, the conversion of 4-hydroxycyclophosphamide to aldophosphamide is rate limiting and is subject to bifunctional catalysis; this reaction can proceed efficiently only in the presence of a bifunctional catalyst. Assuming that the oncotoxic specificity of cyclophosphamide resides with 4-hydroxycyclophosphamide and that its cytotoxic effect at therapeutic doses is largely mediated by phosphoramide mustard released within cells, these observations offer the possibility that the intracellular concentration of bifunctional catalysts, whether in the form of inorganic phosphates, organic phosphates, enzymes, or other species, serve as important determinants with regard to the oncotoxic potential and specificity of cyclophosphamide. Similarly, the concentration of bifunctional catalysis in the urine as well as the pH of the urine may be important with regard to the potential of cyclophosphamide to induce, via acrolein, hemorrhagic cystitis.

Acrolein↗
PubMed1996

A theoretical study of torsional flexibility in the active site of aspartic proteinases: implications for catalysis.

We have performed ab initio Hartree-Fock self-consistent field calculations on the active site of endothiapepsin. The active site was modeled as a formic acid/formate anion moiety (representing the catalytic aspartates, Asp-32 and -215) and a bound water molecule. Residues Gly-34, Ser-35, Gly-217, and Thr-218, which all form hydrogen bonds to the active site, were modeled using formamide and methanol molecules. The water molecule, which is generally believed to function as the attacking nucleophile in catalysis, was allowed to bind to the active site in four distinct configurations. The geometry of each configuration was optimized using two basis sets (4-31G and 4-31G*). The results indicate that in the native enzyme the nucleophilic water is bound in a catalytically inert configuration. However, by rotating the carboxyl group of Asp-32 by about 90 degrees the water molecule can be reorientated to attack the scissile bond of the substrate. A model of the bound enzyme-substrate complex was constructed from the crystal structure of a difluorostatone inhibitor complexed with endothiapepsin. This model suggests that the substrate itself initiates the reorientation of the nucleophilic water immediately prior to catalysis by forcing the carboxyl group of Asp-32 to rotate. The theoretical results predict that the active site of endothiapepsin undergoes a large distortion during substrate binding and this observation has been used to explain some of the kinetics results which have been reported for mutant aspartic proteinases.

Aspartic Acid Endopeptidases↗
PubMed2000

Factors determining the relative stability of anionic tetrahedral complexes in serine protease catalysis and inhibition.

Quantum mechanical ab initio (RHF/6-31+G*//RHF/3-21G) calculations were used to simulate the formation of the tetrahedral complex intermediate (TC) in serine protease active site by substrates and transition-state analog inhibitors. The enzyme active site was simulated by an assembly of the amino acids participating in catalysis, whereas the substrates and inhibitors were simulated by small ligands, acetamide (1) and trifluoroacetone (2), respectively. For the first time, the principal factors determining the relative stability of the TC in serine proteases are arranged according to their energy contributions. These include (a) formation of the new covalent bond between Ser195 O(gamma) and the electrophilic center of a ligand; (b) stabilization of the oxyanion in the oxyanion hole; (c) basic catalysis by His57; and (d) hydrogen bond between Asp102 carboxylate and N(delta) of the protonated His57. We have directly calculated the gas-phase relative free energy of formation of TC(AS)(2) and TC(AS)(1), the value of DeltaDeltaG(g)[TC(AS)(2,1)]. It is DeltaE(cov), the relative energy of the new covalent bond between the enzyme and the ligand formed in a TC that determines the experimentally observed large difference in the stability of TCs formed by substrates and TS-analog inhibitors of serine proteases. We demonstrated that the relative stability of TCs formed by a series of mono- and dipeptide amides and TFKs, derived from experimental kinetic data, can be rather well approximated by the sum of the theoretically calculated value of DeltaDeltaG(g)[TC(AS)(2, 1)] and the difference in hydration free energies of isolated ligands.

Anions↗
PubMed2000

Reactive-site design in folded-polypeptide catalysts--the leaving group pKa of reactive esters sets the stage for cooperativity in nucleophilic and general-acid catalysis.

The second-order rate constants for the hydrolysis of nitrophenyl esters catalysed by a number of folded designed polypeptides have been determined, and 1900-fold rate enhancements over those of the 4-methylimidazole-catalysed reactions have been observed. The rate enhancements are much larger than those expected from the pKa depression of the nucleophilic His residues alone. Kinetic solvent isotope effects were observed at pH values lower than the pKa values of the leaving groups and suggests that general-acid catalysis contributes in the pH range where the leaving group is predominantly protonated. In contrast, no isotope effects were observed at pH values above the pKa of the leaving group. A Hammett rho value of 1.4 has been determined for the peptide-catalysed hydrolysis reaction by variation of the substituents of the leaving phenol. The corresponding values for the imidazole-catalysed reaction is 0.8 and for phenol dissociation is 2.2. There is therefore, very approximately, half a negative charge localised on the phenolate oxygen in the transition state in agreement with the conclusion that transition-state hydrogen-bond formation may contribute to the observed catalysis. The elucidation at a molecular level of the principles that control cooperativity in the biocatalysed ester-hydrolysis reaction represents the first step towards a level of understanding of the concept of cooperativity that may eventually allow us to design tailor-made enzymes for chemical reactions not catalysed by nature.

Amino Acid Sequence↗
PubMed1978

Selective homogenous and heterogeneous catalysis.

Catalysis of organic reactions by micellar aggregates in aqueous solution lacks synthetic utility in its present state, largely for two reasons: first, the selectivity attainable in a loose unstructured micelle is low, with little possibility of attaining the degree of substrate differentiation possible in enzymes, and, second, the scale of reaction is limited, since surfactant must be in large excess over reactant to avoid swamping of the catalytic effect. The discussion will cover initial attempts to overcome these drawbacks. Increased selectivity is apparent in the promotion of ester hydrolysis by micelles of asymmetric chain-functionalized surfactants. In the most favourable circumstances 3:1 discrimination in the rate of reaction of ester enantiomers may be observed. Increased scale of reaction is obtainable when polymer-linked cationic surfactants are used as insoluble catalysts, and these hold promise for various anionactivation reas are used as insoluble catalysts, and these hold promise for varous anion-activation reactions. Specific attention is given to mechanistic aspects of homogenous and heterogeneous catalysis.

Acylation↗
PubMed2004

Palladium oxidase catalysis: selective oxidation of organic chemicals by direct dioxygen-coupled turnover.

Selective aerobic oxidation of organic molecules is a fundamental and practical challenge in modern chemistry. Effective solutions to this problem must overcome the intrinsic reactivity and selectivity challenges posed by the chemistry of molecular oxygen, and they must find application in diverse classes of oxidation reactions. Palladium oxidase catalysis combines the versatility of Pd(II)-mediated oxidation of organic substrates with dioxygen-coupled oxidation of the reduced palladium catalyst to enable a broad range of selective aerobic oxidation reactions. Recent developments revealed that cocatalysts (e.g. Cu(II), polyoxometalates, and benzoquinone) are not essential for efficient oxidation of Pd(0) by molecular oxygen. Oxidatively stable ligands play an important role in these reactions by minimizing catalyst decomposition, promoting the direct reaction between palladium and dioxygen, modulating organic substrate reactivity and permitting asymmetric catalysis.

Alcohols↗
PubMed2006

Signal amplification by allosteric catalysis.

In this article we unify a series of recent studies on bio- and chemosensors under a single signaling strategy: signal amplification by allosteric catalysis (SAAC). The SAAC strategy mimics biological signal transduction processes, where molecular recognition between an external signal and a protein receptor is allosterically transduced into catalytically amplified chemical information (usually second messengers). Several recent biosensing and chemosensing studies apply this nature-inspired strategy by using engineered allosteric enzymes, ribozymes, or regulatable organic catalysts. The factors pertinent to achieving high sensitivity and specificity in SAAC strategies are analyzed. The authors believe that these early studies from a variety of research groups have opened up a new venue for the development of sensing technologies where molecular recognition and catalysis can be coupled for practical purposes.

Allosteric Regulation↗
PubMed2006

Asymmetric heterogeneous catalysis.

Limited natural resources and an increasing demand for enantiomerically pure compounds render catalysis and especially heterogeneous asymmetric catalysis a key technology. The field has rapidly advanced from the initial use of chiral biopolymers, such as silk, as a support for metal catalysts to the modern research areas. Mesoporous supports, noncovalent immobilization, metal-organic catalysts, chiral modifiers: many areas are rapidly evolving. This Review shows that these catalysts have more to them than facile separation or recycling. Better activities and selectivities can be obtained than with the homogeneous catalyst and novel, efficient reaction mechanisms can be employed. Especially fascinating is the outlook for highly ordered metal-organic catalysts that might allow a rational design, synthesis, and the unequivocal structural characterization to give tailor-made catalysts.

Catalysis↗
PubMed2005

Searching sequence space: two different approaches to dihydrofolate reductase catalysis.

There are numerous examples of proteins that catalyze the same reaction while possessing different structures. This review focuses on two dihydrofolate reductases (DHFRs) that have disparate structures and discusses how the catalytic strategies of these two DHFRs are driven by their respective scaffolds. The two proteins are E. coli chromosomal DHFR (Ec DHFR) and a type II R-plasmid-encoded DHFR, typified by R67 DHFR. The former has been described as a very well evolved enzyme with an efficiency of 0.15, while the latter has been suggested to be a model for a "primitive" enzyme that has not yet been optimized by evolution. This comparison underlines what is important to catalysis in these two enzymes and concurrently highlights fundamental issues in enzyme catalysis.

Binding Sites↗
PubMed1994

An RNA chaperone activity of non-specific RNA binding proteins in hammerhead ribozyme catalysis.

We have previously shown that a protein derived from the p7 nucleocapsid (NC) protein of HIV type-1 increases kcat/Km and kcat for cleavage of a cognate substrate by a hammerhead ribozyme. Here we show directly that the increase in kcat/Km arises from catalysis of the annealing of the RNA substrate to the ribozyme and the increase in kcat arises from catalysis of dissociation of the RNA products from the ribozyme. A peptide polymer derived from the consensus sequence of the C-terminal domain of the hnRNP A1 protein (A1 CTD) provides similar enhancements. Although these effects apparently arise from non-specific interactions, not all non-specific binding interactions led to these enhancements. NC and A1 CTD exert their effects by accelerating attainment of the thermodynamically most stable species throughout the ribozyme catalytic cycle. In addition, NC protein is shown to resolve a misfolded ribozyme-RNA complex that is otherwise long lived. These in vitro results suggest that non-specific RNA binding proteins such as NC and hnRNP proteins may have a biological role as RNA chaperones that prevent misfolding of RNAs and resolve RNAs that have misfolded, thereby ensuring that RNA is accessible for its biological functions.

Amino Acid Sequence↗
PubMed1992

Ultrafiltration membranes for the separation of catalyst and products in homogeneous catalysis systems.

Results of preliminary studies on concentration by ultrafiltration and reuse of a Ru(III)EDTA complex in homogeneous catalysis are reported. A method of preparation and selection of conditions of making cellulose acetate membranes for concentrating Ru(III)EDTA is described. At an operating pressure of 75 psig (517 kPa), the above catalyst can be concentrated to seven to eight times its initial concentration. The membrane used exhibits a separation of about 97.5% for the complex with a permeate flow rate of about 3-6 gallons per sq. ft per day. The catalyst activities were determined for two reactions, viz. hydroformylation of 1-hexene and carbonylation of ammonia for five cycles of concentration followed by reuse. The experimental data indicated that the catalyst concentration by ultrafiltration for subsequent reuse is simple and attractive and can provide potentially energy saving alternative to the cumbersome conventional separation processes associated with the recovery of catalysts in homogeneous catalysis.

Biotechnology↗
PubMed1994

Malate dehydrogenase: a model for structure, evolution, and catalysis.

Malate dehydrogenases are widely distributed and alignment of the amino acid sequences show that the enzyme has diverged into 2 main phylogenetic groups. Multiple amino acid sequence alignments of malate dehydrogenases also show that there is a low degree of primary structural similarity, apart from in several positions crucial for nucleotide binding, catalysis, and the subunit interface. The 3-dimensional structures of several malate dehydrogenases are similar, despite their low amino acid sequence identity. The coenzyme specificity of malate dehydrogenase may be modulated by substitution of a single residue, as can the substrate specificity. The mechanism of catalysis of malate dehydrogenase is similar to that of lactate dehydrogenase, an enzyme with which it shares a similar 3-dimensional structure. Substitution of a single amino acid residue of a lactate dehydrogenase changes the enzyme specificity to that of a malate dehydrogenase, but a similar substitution in a malate dehydrogenase resulted in relaxation of the high degree of specificity for oxaloacetate. Knowledge of the 3-dimensional structures of malate and lactate dehydrogenases allows the redesign of enzymes by rational rather than random mutation and may have important commercial implications.

Amino Acid Sequence↗