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At least 145 records · Page 8Linked to original sources
PubMed1992

Catalysis by mutants of human carbonic anhydrase II: effects of replacing hydrophobic residues 198 and 204.

Previous studies shows that the replacement of Phe-198 in carbonic anhydrase III to the corresponding Leu residue found in carbonic anhydrase II caused the appearance of isozyme II-like activity (LoGrasso et al. (1991) Biochemistry 30, 8463-8470). Carbonic anhydrase II is more efficient in the catalysis of CO2 hydration by 500-fold and has an apparent pKa for this catalysis about two pKa units above that of carbonic anhydrase III. Moreover, isozyme II catalyzes the hydrolysis of 4-nitrophenyl acetate, whereas isozyme III shows no appreciable catalysis. The purpose of this work was to test the hypothesis that making the converse replacement Leu-198-->Phe as well as Leu-204-->Glu and the double replacement in carbonic anhydrase II would give the resulting mutants of isozyme II properties of isozyme III. The catalytic activities of these mutants in CO2 hydration and 4-nitrophenyl acetate hydrolysis were smaller by at most 5-fold and the pKa values for these catalyses were identical compared with wild-type isozyme II. The different effects of converse mutants of HCA II and III indicate complexity in structure not evident from their similar backbone conformations.

Base Sequence↗
PubMed2005

Role of cytochrome b5 in catalysis by cytochrome P450 2B4.

Cytochrome b5 has been shown to stimulate, inhibit or have no effect on catalysis by P450 cytochromes. Its action is known to depend on the isozyme of cytochrome P450, the substrate, and experimental conditions. Cytochrome P450 2B4 (CYP 2B4) has been used in our laboratory as a model isozyme to study the role of cytochrome b5 in cytochrome P450 catalysis using two substrates, methoxyflurane and benzphetamine. One substrate is the volatile anesthetic, methoxyflurane, whose metabolism is consistently markedly stimulated by cytochrome b5. The other is benzphetamine, whose metabolism is minimally modified by cytochrome b5. Determination of the stoichiometry of the metabolism of both substrates showed that the amount of product formed is the net result of the simultaneous stimulatory and inhibitory actions of cytochrome b5 on catalysis. Site-directed mutagenesis studies revealed that both cytochrome b5 and cytochrome P450 reductase interact with cytochrome P450 on its proximal surface on overlapping but non-identical binding sites. Comparison of the rate of reduction of oxyferrous CYP 2B4 and the rate of substrate oxidation by cyt b5 and reductase with stopped-flow spectrophotometric and rapid chemical quench experiments has demonstrated that although cytochrome b5 and reductase reduce oxyferrous CYP 2B4 at the same rate, substrate oxidation proceeds more slowly in the presence of the reductase.

Animals↗
PubMed2006

Tertiary contacts distant from the active site prime a ribozyme for catalysis.

Minimal hammerhead ribozymes have been characterized extensively by static and time-resolved crystallography as well as numerous biochemical analyses, leading to mutually contradictory mechanistic explanations for catalysis. We present the 2.2 A resolution crystal structure of a full-length Schistosoma mansoni hammerhead ribozyme that permits us to explain the structural basis for its 1000-fold catalytic enhancement. The full-length hammerhead structure reveals how tertiary interactions occurring remotely from the active site prime this ribozyme for catalysis. G-12 and G-8 are positioned consistent with their previously suggested roles in acid-base catalysis, the nucleophile is aligned with a scissile phosphate positioned proximal to the A-9 phosphate, and previously unexplained roles of other conserved nucleotides become apparent within the context of a distinctly new fold that nonetheless accommodates the previous structural studies. These interactions permit us to explain the previously irreconcilable sets of experimental results in a unified, consistent, and unambiguous manner.

Animals↗
PubMed2001

Catalysis of dehydrogenation of 4-trans-(N,N-dimethylamino)cinnamaldehyde by aldehyde dehydrogenase.

4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA) is a chromophoric and fluorogenic substrate of aldehyde dehydrogenase. Fluorescence of DACA is enhanced by binding to aldehyde dehydrogenase in the absence of catalysis both in the presence and absence of the coenzyme analogue 5'AMP. DACA binds to aldehyde dehydrogenase with a dissociation constant of 1-3 microM and stoichiometry of 2 mol mol(-1) enzyme. Incorporation of DACA during catalysis was also investigated and found to be 2 mol DACA mol(-1) enzyme. Effect of pH on the stoichiometry of DACA incorporation during catalysis has shown that DACA incorporation remained constant at 2 mol DACA mol(-1) enzyme, despite a 74-fold velocity enhancement between pH 5.0 and 9.0. Increase of pH increased decomposition of enzyme-acyl intermediate without affecting the rate-limiting step of the reaction. At pH 7.0 the pH stimulated velocity enhancement was 10-fold over that at pH 5.0; further velocity enhancement (11.5-fold that of pH 7.0) was achieved by 150 microM Mg(2+) ions. The velocity at pH 7.0 with Mg(2+) exceeded that of pH 9.0, and that at maximal pH stimulation at pH 9.5. It was observed that level of intermediate decreased to about 1 mol mol(-1) enzyme, indicating that Mg(2+) ions increased the rate of decomposition of the enzyme-acyl intermediate and shifted the rate-limiting step of the reaction to another step in the reaction sequence.

Aldehyde Dehydrogenase↗
PubMed2002

Enzymatic catalysis in crystals of Escherichia coli maltodextrin phosphorylase.

The bacterial enzyme maltodextrin phosphorylase (MalP) catalyses the phosphorolysis of an alpha-1,4-glycosidic bond in maltodextrins, removing the non-reducing glucosyl residues of linear oligosaccharides as glucose-1-phosphate (Glc1P). In contrast to the well-studied muscle glycogen phosphorylase (GP), MalP exhibits no allosteric properties and has a higher affinity for linear oligosaccharides than GP. We have used MalP as a model system to study catalysis in the crystal in the direction of maltodextrin synthesis. The 2.0A crystal structure of the MalP/Glc1P binary complex shows that the Glc1P substrate adopts a conformation seen previously with both inactive and active forms of mammalian GP, with the phosphate group not in close contact with the 5'-phosphate group of the essential pyridoxal phosphate (PLP) cofactor. In the active MalP enzyme, the residue Arg569 stabilizes the negative-charged Glc1P, whereas in the inactive form of GP this key residue is held away from the catalytic site by loop 280s and an allosteric transition of the mammalian enzyme is required for activation. The comparison between MalP structures shows that His377, through a hydrogen bond with the 6-hydroxyl group of Glc1P substrate, triggers a conformational change of the 380s loop. This mobile region folds over the catalytic site and contributes to the specific recognition of the oligosaccharide and to the synergism between substrates in promoting the formation of the MalP ternary complex. The structures solved after the diffusion of oligosaccharides (either maltotetraose, G4 or maltopentaose, G5) into MalP/Glc1P crystals show the formation of phosphate and elongation of the oligosaccharide chain. These structures, refined at 1.8A and at 2.2A, confirm that only when an oligosaccharide is bound to the catalytic site will Glc1P bend its phosphate group down so it can contact the PLP 5' phosphate group and promote catalysis. The relatively large oligosaccharide substrates can diffuse quickly into the MalP/Glc1P crystals and the enzymatic reaction can occur without significant crystal damage. These structures obtained before and after catalysis have been used as frames of a molecular movie. This movie reveals the relative positions of substrates in the catalytic channel and shows a minimal movement of the protein, involving mainly Arg569, which tracks the substrate phosphate group.

Catalysis↗
PubMed1999

HIV-1 integrase: structural organization, conformational changes, and catalysis.

Integrase comprises three domains capable of folding independently and whose three-dimensional structures are known. However, the manner in which the N-terminal, catalytic core, and C-terminal domains interact in the holoenzyme remains obscure. Catalytically active recombinant IN can exist in a dynamic equilibrium of monomers, dimers, tetramers, and higher order species. Numerous studies indicate that the enzyme functions as a multimer, minimally a dimer. The IN proteins from HIV-1 and ASV have been studied most carefully with respect to the structural basis of catalysis. Although the active site of ASV IN does not undergo significant conformational changes on binding the required metal cofactor, that of HIV-1 IN does. The reversible, metal-induced conformational change in HIV-1 IN impairs the binding of some anti-HIV-1 IN monoclonal antibodies to the enzyme and results in differential susceptibility of the protein to proteolysis. This active site-mediated conformational change reorganizes the catalytic core and C-terminal domains and appears to promote an interaction that is favorable for catalysis. Other metal-dependent structural changes in HIV-1 IN include the promotion of interactions between the N terminal and the catalytic core domains and the induction of tetramers by zinc ions. The end result of these metal-induced changes is apparently the induction of an activated holoenzyme that can form a stable ternary integrase-metal-DNA complex. These structural changes, which appear to be crucial for optimum catalysis in HIV-1 IN, do not occur in ASV IN. The structural changes observed in HIV-1 IN may serve to recruit the catalytic machinery in this enzyme to a conformation that is native for ASV IN.

Animals↗
PubMed2001

Duplication and extension of the Thorneley and Lowe kinetic model for Klebsiella pneumoniae nitrogenase catalysis using a MATHEMATICA software platform.

The Thorneley and Lowe kinetic model for nitrogenase catalysis was developed in the early to mid 1980s, and has been of value in accounting for many aspects of nitrogenase catalysis. It has also been of value by providing a model for predicting new catalytic behavior. Since its original publication, new results have been obtained and have been successfully incorporated into the model. However, the computer program used for nitrogenase simulations has not been generally available. Using kinetic schemes and assumptions previously outlined by Thorneley and Lowe, we report attempts to duplicate the original T&L kinetic simulation for Klebsiella pneumoniae nitrogenase catalysis using an updated simulation based on the MATHEMATICA programming format, which makes it more user-friendly and more readily available. Comparisons of our simulations with the original T&L simulations are generally in agreement, but in some cases serious discrepancy is observed. Possible reasons for the differences are discussed. In addition to duplicating the original T&L model, we report effects of updating it by including information that has come to light subsequent to its original publication.

Adenosine Triphosphate↗
PubMed2002

Dynamic kinetic resolutions and asymmetric transformations by enzymes coupled with metal catalysis.

The combination of enzyme and metal catalysis is described as a useful method for the synthesis of optically active compounds. A key feature of this new methodology is the use of metal catalysts for the in situ racemization of enzymatically unreactive enantiomers in the enzymatic resolution of racemic substrates. So far, two combinations - lipase-ruthenium and lipase-palladium - have been developed for the efficient dynamic kinetic resolution of alcohols and amines. The use of these combinations has also been extended to catalysis of the asymmetric transformation of ketones, their enol acetates, and ketoximes. In most cases, enzyme-metal combination catalysis has provided good yields and high optical purities.

Acetates↗
PubMed2003

The origins of RNA catalysis in ribozymes.

The discovery of RNA catalysis provided a paradigm shift in biology, insight into the evolution of life on the planet and a challenge to understand its mechanistic origins. RNA has limited catalytic resources that must be used to maximal effect. Consequently, RNA catalysis tends to be multifactorial, with several processes contributing to an overall significant enhancement of reaction rate. These include general acid-base catalysis, electrostatic effects, and substrate orientation and proximity. The main players are the RNA nucleobases and bound metal ions. Although most ribozymes carry out phosphoryl transfer, the same considerations appear to apply to peptidyl transfer in the ribosome.

Animals↗
PubMed1995

Trypsin specificity increased through substrate-assisted catalysis.

Histidine 57 of the catalytic triad of trypsin was replaced with alanine to determine whether the resulting variant would be capable of substrate-assisted catalysis [Carter, P., & Wells, J. A. (1987) Science 237, 394-9]. A 2.5-fold increase in kcat/Km was observed on tri- or tetrapeptide substrates containing p-nitroanilide leaving groups and histidine at P2. In contrast, hydrolysis of peptide substrates extending from P6 to P6' is improved 70-300-fold by histidine in the P2 or P1' position. This preference creates new protease specificities for sequences HR decreases, R decreases H, HK decreases, and K decreases H. The ability of histidine from either the P2 or the P1' position of substrate to participate in catalysis emphasizes the considerable variability of proteolytically active orientations which can be assumed by the catalytic triad. Trypsin H57A is able to hydrolyze fully folded ornithine decarboxylase with complete specificity at a site containing the sequence HRH. Trypsin H57A was compared to enteropeptidase in its ability to cleave a propeptide from trypsinogen. Trypsin H57A cleaved the propeptide of a variant trypsinogen containing an introduced FPVDDDHR cleavage site only 100-fold slower than enteropeptidase cleaved trypsinogen. The selective cleavage of folded proteins suggests that trypsin H57A can be used for specific peptide and protein cleavage. The extension of substrate-assisted catalysis to the chymotrypsin family of proteolytic enzymes indicates that it may be possible to apply this strategy to a wide range of serine proteases and thereby develop various unique specificities for peptide and protein hydrolysis.

Amino Acid Sequence↗
PubMed2000

Histidine-607 and histidine-643 provide important interactions for metal support of catalysis in phosphodiesterase-5.

Class I cyclic nucleotide phosphodiesterases (PDEs) share a catalytic domain containing 18 invariant residues. In cGMP-binding cGMP-specific PDE (PDE5), we showed previously that point mutation of nine of these profoundly decreases k(cat) when the assay is conducted in the presence of Mg(2+); seven of these are in the prototypical metal-binding motifs A and B (HX(3)HX(n)()E) that we identified earlier. Tandem arrangement of two of these metal-binding motifs in PDEs is novel, and whether residues within these motifs are involved in metal support of catalytic activity is a fundamental question in this field. This report shows that mutation of either His-607 (A motif) or His-643 (B motif) to alanine profoundly diminishes support of PDE catalysis by Mn(2+) or Mg(2+), but mutation of His-647 in B motif or of Glu in either motif does not. H607A and H643A mutants have much greater maximum catalytic rates supported by Mn(2+) than that by Mg(2+); catalytic activity of H603A mutant is supported weakly by either. In H607A and H643A, K(a)s for Mn(2+) and Mg(2+) are increased, but the effect of Mn(2+) is 2-fold greater than that of Mg(2+) in each. Mutation of any of the other conserved residues (Asn-604, Asp-644, His-675, Asp-714, and Asp-754) causes unremarkable changes in Mn(2+) or Mg(2+) support of catalysis. This study identifies specific residues in PDE5 that contribute to interactions with catalytically relevant metals. The combined data suggest that despite a high degree of sequence similarity between each HX(3)HX(n)()E motif in PDEs and certain metallo-endopeptidases, PDEs employ a distinct complement of residues for interacting with metals involved in catalysis.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1995

Solvent-derived protons in catalysis by brewers' yeast pyruvate decarboxylase.

Catalysis of proton transfer to thiamin diphosphate (TDP) and 2-(1-hydroxyethyl)thiamin diphosphate (HETDP) by pyruvate decarboxylase isozymes (PDC; EC 4.1.1.1) from Saccharomyces carlsbergensis was investigated by determining the solvent discrimination tritium isotope effect, (kH/kT)disc, on the reaction of pyruvate to form acetaldehyde in the presence of the nonsubstrate allosteric effector pyruvamide. The fractionation factors for TDP C(2)-L (phi C(2) = 0.98 +/- 0.06) and HETDP C(alpha)-L (phi C(alpha) = 1.01 +/- 0.07) (L = H or D) do not contribute significantly to observed enzymic isotopic discrimination. The value of (kH/kT)disc = 1.0 for reprotonation of TDP C(2)-L under single-turnover conditions ([E] > [S]) is consistent with C(2)-hydron transfer via a catalytic group (phi = 1) equilibrated with solvent. [1-L]Acetaldehyde formation under transient steady-state ([E] < [S]) conditions shows solvent discrimination tritium isotope effects that increase over the range (kH/kT)disc = 0.39 (single turnover) to 0.86 (ten turnovers). The 2-fold increase in the value of (kH/kT)disc for the [1-L]acetaldehyde product under steady-state compared to single-turnover conditions is attributed to a fractionation factor of phi 1 = 0.88 +/- 0.06 for the residue(s) involved in C(alpha)-hydron transfer to form HETDP. This provides evidence that catalysis of acetaldehyde formation by PDC involves specific protonation of both HETDP C(alpha)-L and TDP C(2)-L (phi 2 = 1.0 +/- 0.1) and requires at least two catalytic groups. Values of phi < or = 1 for protonation of TDP C(2)-L and HETDP C(alpha)-L provide no evidence that the exocyclic 4'-amino or -imino group (phi > or = 1.2) provides significant intramolecular catalysis in the enzyme-bound coenzyme.

Acetaldehyde↗
PubMed1991

Analysis of hydride transfer and cofactor fluorescence decay in mutants of dihydrofolate reductase: possible evidence for participation of enzyme molecular motions in catalysis.

A remarkable correlation has been discovered between fluorescence lifetimes of bound NADPH and rates of hydride transfer among mutants of dihydrofolate reductase (DHFR) from Escherichia coli. Rates of hydride transfer from NADPH to dihydrofolate change by a factor of 1,000 for the series of mutant enzymes. Since binding constants for the initial complex between coenzyme and DHFR change by only a factor of 10, the major portion of the change in hydride transfer must be attributed to losses in transition-state stabilization. The time course of fluorescence decay for NADPH bound to DHFR is biphasic. Lifetimes ranging from 0.3 to 0.5 ns are attributed to a solvent-exposed dihydronicotinamide conformation of bound coenzyme which is presumably not active in catalysis, while decay times (tau 2) in the range of 1.3 to 2.3 ns are assigned to a more tightly bound species of NADPH in which dihydronicotinamide is sequestered from solvent. It is this slower component that is of interest. Ternary complexes with three different inhibitors, methotrexate, 5-deazafolate, and trimethoprim, were investigated, along with the holoenzyme complex; 3-acetylNADPH was also investigated. Fluorescence polarization decay, excitation polarization spectra, the temperature variation of fluorescence lifetimes, fluorescence amplitudes, and wavelength of absorbance maxima were measured. We suggest that dynamic quenching or internal conversion promotes decay of the excited state in NADPH-DHFR. When rates of hydride transfer are plotted against the fluorescence lifetime (tau 2) of tightly bound NADPH, an unusual correlation is observed. The fluorescence lifetime becomes longer as the rate of catalysis decreases for most mutants studied. However, the fluorescence lifetime is unchanged for those mutations that principally alter the binding of dihydrofolate while leaving most dihydronicotinamide interactions relatively undisturbed. The data are interpreted in terms of possible dynamic motions of a flexible loop region in DHFR which closes over both substrate and coenzyme binding sites. These motions could lead to faster rates of fluorescence decay in holoenzyme complexes and, when correlated over time, may be involved in other motions which give rise to enhanced rates of catalysis in DHFR.

Catalysis↗
PubMed2000

Effect of site-directed mutagenesis of the conserved aspartate and glutamate on E. coli undecaprenyl pyrophosphate synthase catalysis.

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes condensation of eight molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate to yield C(55)-undecaprenyl pyrophosphate. We have mutated the aspartates and glutamates in the five conserved regions (I to V) of UPPs protein sequence to evaluate their effects on substrate binding and catalysis. The mutant enzymes including D26A, E73A, D150A, D190A, E198A, E213A, D218A, and D223A were expressed and purified to great homogeneity. Kinetic analyses of these mutant enzymes indicated that the substitution of D26 in region I with alanine resulted in a 10(3)-fold decrease of k(cat) value compared to wild-type UPPs. Its IPP K(m) value has only minor change. The mutagenesis of D150A has caused a much lower IPP affinity with IPP K(m) value 50-fold larger than that of wild-type UPPs but did not affect the FPP K(m) and the k(cat). The E213A mutant UPPs has a 70-fold increased IPP K(m) value and has a 100-fold decreased k(cat) value compared to wild-type. These results suggest that D26 of region I is critical for catalysis and D150 in region IV plays a significant role of IPP binding. The E213 residue in region V is also important in IPP binding as well as catalysis. Other mutant UPPs enzymes in this study have shown no significant change (<5-fold) of k(cat) with exception of E73A and D218A. Both enzymes have 10-fold lower k(cat) value relative to wild-type UPPs.

Alkyl and Aryl Transferases↗
PubMed1992

The contribution of threonine 55 to catalysis in aspartate transcarbamoylase.

Heavy-atom isotope effects and steady-state kinetic parameters were measured for the catalytic trimer of an active site mutant of aspartate transcarbamoylase, T55A, to assess the role of Thr 55 in catalysis. The binding of carbamoyl phosphate to the T55A mutant was decreased by 2 orders of magnitude relative to the wild-type enzyme whereas the affinities for aspartate and succinate were not markedly altered. This indicates that Thr 55 plays a significant role in the binding of CbmP. If, as had been suggested previously, Thr 55 assists in the polarization of the carbonyl group of CbmP, the carbon isotope effect for the T55A mutant should increase relative to that observed for the wild-type enzyme. However, the opposite is seen, indicating that Thr 55 is not involved in stabilizing the oxyanion in the transition state. Quantitative analysis of a series of 13C and 15N isotope effects suggested that the rate-determining step in the reaction catalyzed by T55A trimer may be a conformational change in the protein subsequent to formation of the Michaelis complex. Thus, Thr 55 may facilitate a conformational change in the enzyme that is a prerequisite for catalysis. An altered active site environment in the binary and Michaelis complexes with T55A trimer is reflected in the pH profiles for log V, log (V/K)asp, and pK(i) succinate, show a displacement in the pK values of ionizing residues involved in aspartate binding and catalysis relative to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartate Carbamoyltransferase↗
PubMed1994

Kinetic evidence for separate site catalysis by pyruvate phosphate dikinase.

Pyruvate phosphate dikinase from Clostridium symbiosum catalyzes the interconversion of adenosine-5'-triphosphate (ATP), orthophosphate (Pi), and pyruvate with adenosine 5'-monophosphate (AMP), inorganic pyrophosphate (PPi), and phosphoenolpyruvate (PEP) using a bi (ATP, Pi) bi (AMP, PPi) uni (pyruvate) uni (PEP) kinetic mechanism and pyrophosphorylenzyme (E-PP) and phosphorylenzyme (E-P) covalent intermediates. The present studies were carried out to determine whether or not the site of catalysis of the E + ATP + P1 E-P + AMP + PPi partial reaction overlaps with that of the E-P + pyruvate E + PEP partial reaction. Single-turnover experiments were carried out to test the effect of binding site occupancy on catalysis at a second site. Saturation of the enzyme with adenyl imidodiphosphate (AMPPNP) inhibited [32P]E-P formation from [beta-32P]ATP and Pi but did not significantly inhibit [32P]E-P formation from [32P]PEP. Likewise, saturation of E-P with AMP did not significantly inhibit [14C]PEP formation from [14C]pyruvate, suggesting separate, largely independent ATP/AMP vs pyruvate/PEP sites. Movement of the phosphorylhistidine residue between reaction sites was probed by testing oxalate as an inhibitor of phosphoryl transfer from E-P to [14C]pyruvate or to [14C]AMP. Both phosphoryl transfers were inhibited. These results were interpreted as evidence for the requirement for phosphorylhistidine release from the pyruvate site prior to participation in catalysis at the nucleotide site.

Adenosine Monophosphate↗
PubMed1994

Phosphatidylinositol-specific phospholipase C from Bacillus cereus at the lipid-water interface: interfacial binding, catalysis, and activation.

Binding characteristics of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus binding to the phospholipid-water interface were determined by spectroscopic methods and correlated with PI-PLC's catalytic properties. Binding of the enzyme to micelles and bilayers of zwitterionic phosphocholines is accompanied by an increase in the fluorescence emission from tryptophan, whereas a decrease in the emission is observed with synthetic anionic lipids containing a phosphomethanol head group. A similar decrease in the tryptophan emission is observed with phosphatidylinositol (PI) analogues containing the phospho-D-1-myo-inositol head group, but not with the enantiomeric L-1-myo-inositol. In covesicles of PI and phosphatidylcholine (PC), the rate of cleavage of PI is reduced because, as a neutral diluent, PC effectively reduces the surface concentration of PI that the bound enzyme "sees" in the interface. This permits determination of the interfacial Michaelis constant (KM*) as 0.26 mol fraction for PI as substrate. On the other hand, ditetradecylglycerophosphomethanol (DTPM) acts as a kinetic competitive inhibitor in the covesicles. The spectroscopic and catalytic activity data taken together show that PI-PLC binds to the interface of aqueous dispersions of phospholipids with an apparent Kd (in terms of the lipid monomers) of about 10-50 microM. However, only lipids with an anionic head group, such as phosphomethanol and phospho-D-1-myo-inositol, are able to bind as single molecules into the active site of the enzyme at the interface. Enantiomeric phospho-L-1-myo-inositol or the zwitterionic phosphocholine head group has little affinity for the enzyme at the interface. Thus, PI-PLC appears to obey the two-stage, Michaelis-Menten adaptation of interfacial catalysis, according to which the binding of the enzyme to the interface precedes the steps of the catalytic turnover at the interface. Limit estimates suggest that on PI or PI/PC vesicles the catalysis occurs in the "scooting" mode with a moderate processivity. DTPM vesicles also inhibit the activity of PI-PLC toward the synthetic water-soluble substrate myo-inositol 1-(4-nitrophenyl phosphate), but the activity is enhanced severalfold in the presence of vesicles of zwitterionic phosphatidylcholine. Several possible explanations of this interfacial activation are considered within the general context of the kinetic scheme for interfacial catalysis. The kinetic results for the action of PI-PLC bound to vesicles are consistent with a model in which the interface acts as an "allosteric" effector of the catalytic rate constant, kcat, without affecting the substrate binding.

Bacillus cereus↗
PubMed1994

Deamidation of triosephosphate isomerase in reverse micelles: effects of water on catalysis and molecular wear and tear.

The specific deamidation of asparagine-71 of triosephosphate isomerase increases upon substrate binding and catalysis. This deamidation at the dimer interface initiates subunit dissociation, unfolding, and protein degradation. The apparent connection between catalysis and terminal marking supports the concept of "molecular wear and tear", and raises questions related to the molecular events that lead to deamidation. In order to explore this interaction, triosephosphate isomerase was entrapped in reverse micelles with different water contents that support different catalytic rates. Deamidation was quantified for the free enzyme, the enzyme in the presence of substrates, and the enzyme which had been covalently modified at the catalytic center with the substrate analogue 3-chloroacetol phosphate (CAP). Both in water and in reverse micelles of cetyltrimethylammonium with 3% and 6% water, substrate binding enhanced deamidation. Studies of the extent of deamidation at various water concentrations showed that deamidation per catalytic turnover was about 6 and 17 times higher in 6% and 3% water than in 100% water, respectively. The enzyme was also entrapped in micelles formed with toluene, phospholipids, and Triton X-100 to explore the process at much lower water concentrations (e.g., 0.3%). Under these conditions, catalysis was very low, and hardly any deamidation took place. Deamidation of the CAP-labeled enzyme was also markedly diminished. At these low-water conditions, the enzyme exhibited markedly increased thermostability and resistance to hydrolysis of the amide bonds. The data suggest that the rate of deamidation not only is dependent on the number of catalytic events but also is related to the time that asparagine-71 exists in a conformation or solvent environment more favorable for deamidation.

Amides↗