Increase In Kcat Research Articles

The Acidic COOH-Terminal Region of Factor Va Heavy Chain Controls the Rate of Prothrombin Activation by Prothrombinase.

The blood coagulation cascade is initiated at the site of vascular injury and results in the activation of prothrombin (Pro) to thrombin by the prothrombinase (IIase) complex. IIase is composed of the enzyme, factor Xa (fXa) bound to its cofactor, factor Va (fVa) on a phospholipid surface in the presence of Ca2+ ions. Two pathways for Pro activation are possible: membrane-bound fXa alone activates Pro following initial cleavage at Arg271 followed by cleavage at Arg320, while the fully assembled IIase activates Pro following the opposite pathway, initial cleavage at Arg320 followed by cleavage at Arg271. Activation of Pro via this latter pathway is characterized by the generation of a transient intermediate, meizothrombin (MzT) that has proteolytic activity. Initial cleavage of prothrombin at Arg320 resulting in MzT generation is absolutely fVa-dependent. Human factor V (fV) is activated by thrombin to produce a heterodimer consisting of a heavy chain and a light chain associated through divalent metal ions. The heavy chain of fVa contains an acidic hirudin-like region at the COOH-terminus (amino acids 680–709). We have shown using overlapping peptides from this region that a pentapeptide with the sequence DYDYQ inhibits Pro activation by IIase by inhibiting MzT generation. In has been reported that various proteases can cleave the acidic region of fVa heavy chain to produce a cofactor with a truncated heavy chain. All the studies revealed that removal of the acidic COOH-terminal portion of fVa heavy chain results in a cofactor molecule that is deficient in its clotting activity in a clotting assay using fV-deficient plasma, however IIase assembled with cofactor molecules missing the acidic COOH-terminus have significant higher kcat for Pro activation as assessed in an assay using purified reagents and a chromogenic substrate specific for thrombin. A molecular explanation for these paradoxical observations has not yet been provided. We have created a mutant recombinant fV molecule that is missing the last 30 amino acids from the heavy chain (fVΔ680-709). The clotting activity of the mutant molecule was impaired as compared to wild type fVa. IIase assembled with fVaΔ680-709 demonstrated a 30–40% increase in the kcat for the activation of Pro. Interstingly, gel electrophoresis revealed a delay in Pro activation with persistence of MzT during activation. Further experiments demonstrated that peptide DYDYQ inhibited MzT formation by IIase assembled with fVaΔ680-709. It has been well established that while MzT has poor clotting activity, its amidolytic activity is considerable increased towards small chromogenic substrates compared to thrombin. A logical explanation that will reconcile all the findings described above is that, the acidic COOH-terminus of fVa heavy chain regulates MzT concentration within IIase during the factor Xa catalyzed Pro activation. Thus, activation of Pro by IIase assembled with a cofactor that is missing the acidic region will result in increased MzT production. This result will be translated by a fVa molecule that is deficient in its clotting activity and produces an increase in kcat when introduced into IIase. In contrast, in the presence of an excess of DYDYQ no MzT is made by IIase resulting in the slow generation of thrombin through the alternative pathway. Our data are consistent with the interpretation that the acidic COOH-terminus of fVa heavy chain contributes a major productive interactive site for Pro within IIase.

Transition-state variation in human, bovine, and Plasmodium falciparum adenosine deaminases.

Adenosine deaminases (ADAs) from human, bovine, and Plasmodium falciparum sources were analyzed by kinetic isotope effects (KIEs) and shown to have distinct but related transition states. Human adenosine deaminase (HsADA) is present in most mammalian cells and is involved in B- and T-cell development. The ADA from Plasmodium falciparum (PfADA) is essential in this purine auxotroph, and its inhibition is expected to have therapeutic effects for malaria. Therefore, ADA is of continuing interest for inhibitor design. Stable structural mimics of ADA transition states are powerful inhibitors. Here we report the transition-state structures of PfADA, HsADA, and bovine ADA (BtADA) solved using competitive kinetic isotope effects (KIE) and density functional calculations. Adenines labeled at [6-13C], [6-15N], [6-13C, 6-15N], and [1-15N] were synthesized and enzymatically coupled with [1'-14C] ribose to give isotopically labeled adenosines as ADA substrates for KIE analysis. [6-13C], [6-15N], and [1-15N]adenosines reported intrinsic KIE values of (1.010, 1.011, 1.009), (1.005, 1.005, 1.002), and (1.004, 1.001, 0.995) for PfADA, HsADA, and BtADA, respectively. The differences in intrinsic KIEs reflect structural alterations in the transition states. The [1-15N] KIEs and computational modeling results indicate that PfADA, HsADA, and BtADA adopt early SNAr transition states, where N1 protonation is partial and the bond order to the attacking hydroxyl nucleophile is nearly complete. The key structural variation among PfADA, HsADA, and BtADA transition states lies in the degree of N1 protonation with the decreased bond lengths of 1.92, 1.55, and 1.28 A, respectively. Thus, PfADA has the earliest and BtADA has the most developed transition state. This conclusion is consistent with the 20-36-fold increase of kcat in comparing PfADA with HsADA and BtADA.

The Molecular Basis of Glyphosate Resistance by an Optimized Microbial Acetyltransferase

GAT is an N-acetyltransferase from Bacillus licheniformis that was optimized by gene shuffling for acetylation of the broad spectrum herbicide, glyphosate, forming the basis of a novel mechanism of glyphosate tolerance in transgenic plants (Castle, L. A., Siehl, D. L., Gorton, R., Patten, P. A., Chen, Y. H., Bertain, S., Cho, H. J., Duck, N., Wong, J., Liu, D., and Lassner, M. W. (2004) Science 304, 1151-1154). The 1.6-A resolution crystal structure of an optimized GAT variant in ternary complex with acetyl coenzyme A and a competitive inhibitor, 3-phosphoglyerate, defines GAT as a member of the GCN5-related family of N-acetyltransferases. Four active site residues (Arg-21, Arg-73, Arg-111, and His-138) contribute to a positively charged substrate-binding site that is conserved throughout the GAT subfamily. Structural and kinetic data suggest that His-138 functions as a catalytic base via substrate-assisted deprotonation of the glyphosate secondary amine, whereas another active site residue, Tyr-118, functions as a general acid. Although the physiological substrate is unknown, native GAT acetylates D-2-amino-3-phosphonopropionic acid with a kcat/Km of 1500 min-1 mM-1. Kinetic data show preferential binding of short analogs to native GAT and progressively better binding of longer analogs to optimized variants. Despite a 200-fold increase in kcat and a 5.4-fold decrease in Km for glyphosate, only 4 of the 21 substitutions present in R7 GAT lie in the active site. Single-site revertants constructed at these positions suggest that glyphosate binding is optimized through substitutions that increase the size of the substrate-binding site. The large improvement in kcat is likely because of the cooperative effects of additional substitutions located distal to the active site.

Binding of Elongation Factor eEF1A2 to Phosphatidylinositol 4-Kinase β Stimulates Lipid Kinase Activity and Phosphatidylinositol 4-Phosphate Generation

Eukaryotic protein translation elongation factor 1 alpha 2 (eEF1A2) is an oncogene that transforms mammalian cell lines and increases their tumorigenicity in nude mice. Increased expression of eEF1A2 occurs during the development of breast, ovarian, and lung cancer. Here, we report that eEF1A2 directly binds to and activates phosphatidylinositol 4-kinase III beta (PI4KIIIbeta), an enzyme that converts phosphatidylinositol to phosphatidylinositol 4-phosphate. Purified recombinant eEF1A2 increases PI4KIIIbeta lipid kinase activity in vitro, and expression of eEF1A2 in rat and human cells is sufficient to increase overall cellular phosphatidylinositol 4-kinase activity and intracellular phosphatidylinositol 4-phosphate abundance. siRNA-mediated reduction in eEF1A2 expression concomitantly reduces phosphatidylinositol 4-kinase activity. This identifies a physical and functional relationship between eEF1A2 and PI4KIIIbeta.

Characterization of an Ideal Amphipathic Peptide as a Potential Hemostatic Agent.

We have previously demonstrated that amphipathic helical peptides can accelerate the turnover of the substrate factor X by the enzyme, factor IXa in a gamma-carboxyglutamic acid (Gla) domain dependent manner (Biochemistry,39:12000Biochemistry,39:12000). Such improvement was due to a remarkable decrease in KM and a mild increase in kcat, mimicking the presence of phospholipid membranes. It is hypothesized that amphipathic helical peptides could exert similar activities in other reactions of the blood coagulation cascade, such as thrombin generation and in whole blood clotting. In the present work, we analyze the activity of a 22-amino acid ideal amphipathic helical peptide (IAP) of K7L15 composition, in factor X activation and thrombin generation. The addition of IAP accelerates factor X activation by factor IXa in a concentration dependent manner. IAP also accelerates thrombin generation by factor Xa with a comparable peptide and substrate concentration dependence. Further, the Gla domain is also required for peptide activity confirming the hypothesis this peptide behaves as a membrane mimetic. Using fluorescence spectroscopy and an ELISA-based binding assay, we demonstrate direct binding between IAP and the Gla domain of factor X. Additionally, when IAP is immobilized to a reaction surface, factor X activation and thrombin generation are dramatically enhanced, as compared to the corresponding reactions on untreated surfaces. Finally, activated partial thromboplastin times (APTTs) of pooled plasma are significantly shortened on surfaces treated with IAP, in comparison with clotting times measured on untreated surfaces. The above results suggest that immobilized IAP may serve as a potential hemostatic agent, as demonstrated in isolated critical reactions of the coagulation cascade and in whole plasma.

Kosmotropic Salt Activation and Substrate Specificity of Poliovirus Protease 3C

Picornaviruses produce a large polyprotein, which is cleaved by virally encoded cysteine peptidases, picornain-2A and -3C. Picornain-3C has characteristics of both the serine peptidase chymotrypsin and the cysteine peptidase papain in that the 3D structure resembles chymotrypsin, but its nucleophile is a cysteine SH rather than a serine OH group. We investigated the specificity of poliovirus picornain-3C (PV3C) protease and the influence of kosmotropic salts on catalytic activity, using FRET peptides related to a cleavable segment of the virus polyprotein. The peptidase activity of PV3C was found to be 100-fold higher in the presence of 1.5 M sodium citrate. This activation was anion-dependent, following the Hofmeister series citrate(3-) > SO4(2-) > HPO4(2-) > acetate- > HCO3(-) > Cl-. The activation appeared to be independent of substrate sequence and arose primarily from an increase in kcat. A shift to higher pH was also observed for the pK1 of the enzyme pH-activity profile. Experiments with the fluorescent probe ANS (1-anilino-8-naphthalene sulfonate) showed that the protease bound the dye in the presence of 1 M sodium citrate but not in its absence or in the presence of 1 M NaCl. Structural changes in PV3C protease were detected using circular dichroism and the thermodynamic data indicated a more organized active site in the presence of sodium citrate. PV3C protease was also activated in D2O, which was added to the activation by citrate. These effects seem to be related to nonspecific interactions between the solvent and the protein. Our data show that the catalytic efficiency of PV3C protease is modulated by the composition of the environment and that this modulation may play a role in the optimal processing of polyprotein for the virus assembly that occurs inside specific vesicles formed in poliovirus-infected cells.

Evolution of Enzymatic Activity in the Tautomerase Superfamily: Mechanistic and Structural Consequences of the L8R Mutation in 4-Oxalocrotonate Tautomerase,

4-Oxalocrotonate tautomerase (4-OT) and trans-3-chloroacrylic acid dehalogenase (CaaD) are members of the tautomerase superfamily, a group of structurally homologous proteins that share a beta-alpha-beta fold and a catalytic amino-terminal proline. 4-OT, from Pseudomonas putida mt-2, catalyzes the conversion of 2-oxo-4-hexenedioate to 2-oxo-3-hexenedioate through the dienol intermediate 2-hydroxymuconate in a catabolic pathway for aromatic hydrocarbons. CaaD, from Pseudomonas pavonaceae 170, catalyzes the hydrolytic dehalogenation of trans-3-chloroacrylate in the trans-1,3-dichloropropene degradation pathway. Both reactions may involve an arginine-stabilized enediolate intermediate, a capability that may partially account for the low-level CaaD activity of 4-OT. Two active-site residues in 4-OT, Leu-8 and Ile-52, have now been mutated to the positionally conserved and catalytic ones in CaaD, alphaArg-8, and alphaGlu-52. The L8R and L8R/I52E mutants show improved CaaD activity (50- and 32-fold increases in k(cat)/K(m), respectively) and diminished 4-OT activity (5- and 1700-fold decreases in k(cat)/K(m), respectively). The increased efficiency of L8R-4-OT for the CaaD reaction stems primarily from an 8.8-fold increase in k(cat), whereas that of the L8R/I52E mutant is due largely to a 23-fold decrease in K(m). The presence of the additional arginine residue in the active site of L8R-4-OT does not alter the pK(a) of the Pro-1 amino group from that measured for the wild type (6.5 +/- 0.1 versus 6.4 +/- 0.2). Moreover, the crystal structure of L8R-4-OT is comparable to that of the wild type. Hence, the enhanced CaaD activity of L8R-4-OT is likely due to the additional arginine residue that can participate in substrate binding and/or stabilization of the putative enediolate intermediate. The results also suggest that the evolution of new functions within the tautomerase superfamily could be quite facile, requiring only a few strategically placed active-site mutations.

Regulation of prokaryotic adenylyl cyclases by CO2

The Slr1991 adenylyl cyclase of the model prokaroyte Synechocystis PCC 6803 was stimulated 2-fold at 20 mM total C(i) (inorganic carbon) at pH 7.5 through an increase in k(cat). A dose response demonstrated an EC50 of 52.7 mM total C(i) at pH 6.5. Slr1991 adenylyl cyclase was activated by CO2, but not by HCO3-. CO2 regulation of adenylyl cyclase was conserved in the CyaB1 adenylyl cyclase of Anabaena PCC 7120. These adenylyl cyclases represent the only identified signalling enzymes directly activated by CO2. The findings prompt an urgent reassessment of the activating carbon species for proposed HCO3--activated adenylyl cyclases.

Differential regulation of glutamate‐cysteine ligase subunit expression and increased holoenzyme formation in response to cysteine deprivation

Glutamate-cysteine ligase (GCL) is a heterodimer of a catalytic subunit (GCLC) that possesses all of the enzymatic activity and a modifier subunit (GCLM) that alters the Ki of GCLC for GSH. We hypothesized that the expression of GCLM and the association of GCLM with GCLC was responsible for the apparent increase in GCL activity state observed in liver of rats fed low protein diets or in hepatocytes cultured in low-sulfur amino acid-containing medium. Therefore, we conducted a series of studies with rats and with a human hepatoma cell line (HepG2/C3A) to assess the role of GCLM and holoenzyme formation in the regulation of GCL activity in response to sulfur amino acid intake or availability. Increases in GCL activity in rat liver, as well as in HepG2 cells, were due to the additive effects of changes in GCLC amount and GCL kcat. The increase in GCL kcat was associated with increased holoenzyme formation, which was associated with an increase in the molar ratio of GCLM to GCLC. Furthermore, our results indicate that the GCLM level in rat liver is always limiting and that up-regulation of the GCLM level results in increased holoenzyme formation and an increase in kcat. This is the first report demonstrating that the catalytic efficiency of rat GCL is increased by holoenzyme formation and the first demonstration of differential up-regulation of the GCL subunits in response to cysteine deprivation.

Biphasic Effect of Simple Phenol Derivatives on Mushroom Tyrosinase Activity

In searching for inhibitors of mushroom tyrosinase, we observed that a number of phenol derivatives showed a biphasic effect on oxidation of the catechol DOPA. These compounds stimulated activity at low concentrations and inhibited at higher concentrations. The stimulatory effect is due to an increase in kcat; the inhibition is due to an increase in Km (and is thus competitive). These compounds show only competitive inhibition when phenol is used as a substrate. The EC50 for compounds acting as activators of catechol oxidation show a strong positive correlation with their EC50 as inhibitors of phenol oxidation. Taken together, these results suggest that mushroom tyrosinase contains two distinct binding sites for phenols: a high affinity site whose occupancy causes allosteric activation of catechol oxidation and competitive inhibition of phenol oxidation, and a second low affinity site whose occupancy causes competitive inhibition of catechol oxidation. The data also suggest that the active site for phenol oxidation may be distinct from the active site for catechol oxidation. Support from the Adrian Tinsley Program for Undergraduate Research is gratefully acknowledged.

Pseudomonas aeruginosa C5-mannuronan epimerase: steady-state kinetics and characterization of the product.

Alginate is a major constituent of mature biofilms produced by Pseudomonas aeruginosa. The penultimate step in the biosynthesis of alginate is the conversion of some beta-D-mannuronate residues in the polymeric substrate polymannuronan to alpha-L-guluronate residues in a reaction catalyzed by C5-mannuronan epimerase. Specificity studies conducted with size-fractionated oligomannuronates revealed that the minimal substrate contained nine monosaccharide residues. The maximum velocity of the reaction increased from 0.0018 to 0.0218 s(-1) as the substrate size increased from 10 to 20 residues, and no additional increase in kcat was observed for substrates up to 100 residues in length. The Km decreased from 80 microM for a substrate containing fewer than 15 residues to 4 microM for a substrate containing more than 100 residues. In contrast to C5-mannuronan epimerases that have been characterized in other bacterial species, P. aeruginosa C5-mannuronan epimerase does not require Ca2+ for activity, and the Ca2+-alginate complex is not a substrate for the enzyme. Analysis of the purified, active enzyme by inductively coupled plasma-emission spectroscopy revealed that no metals were present in the protein. The pH dependence of the kinetic parameters revealed that three residues on the enzyme which all have a pKa of approximately 7.6 must be protonated for catalysis to occur. The composition of the polymeric product of the epimerase reaction was analyzed by 1H NMR spectroscopy, which revealed that tracts of adjacent guluronate residues were readily formed. The reaction reached an apparent equilibrium when the guluronate composition of the polymer was 75%.

Escherichia coli Cyclopropane Fatty Acid Synthase: Is a Bound Bicarbonate Ion the Active-Site Base?

Cyclopropane synthases catalyze the cyclopropanation of unsaturated fatty acid using S-adenosyl-L-methionine as the methylene donor. The crystal structure of three cyclopropane synthases from Mycobacterium tuberculosis showed a bicarbonate ion bound in the active site that was proposed to act as a general base in the reaction mechanism [Huang, C., Smith, V., Glickman, M. S., Jacobs, W. R., and Sacchettini, J. C. (2002) J. Biol. Chem. 277, 11559-11569]. Because the in vitro activity of M. tuberculosis cyclopropane synthases has not yet been reported and because the ligands of the bicarbonate ion are all strictly conserved in cyclopropane synthases, we used the closely related Escherichia coli cyclopropane fatty acid synthase for this study. The putative ligands that share a hydrogen bond with the bicarbonate through their side chains were mutated. H266A, Y317F, E239A, and E239Q mutants were thus constructed and purified, and their catalytic efficiencies were 5.3, 0.7, 0.2, and <0.02%, respectively. C139 that is bound to the bicarbonate by its NH amide had already been mutated to serine in a previous work, and this mutant retains 31% of the activity of the wild-type enzyme. Kinetic analyses and binding studies using spectrofluorimetry showed that these mutations affected the catalytic constant rather than the binding of the substrates. While addition of free bicarbonate had almost no effect on the wild-type enzyme activity, all mutants, with the exception of E239A and E239Q, were rescued by the addition of free bicarbonate. The catalytic efficiencies of the rescued mutants were 85, 16, and 14% for C139S, H266A, and Y317F, respectively. This effect was specific to bicarbonate. The kinetic parameters of the rescued mutants were determined, and it is shown that the rescuing effect is due to an increase in kcat. These data are interpreted by assuming that the E. coli cyclopropane fatty acid synthase specifically binds a bicarbonate ion that is involved in catalysis, as proposed for the M. tuberculosis enzymes, and that mutation of the bicarbonate ligands decreases the affinity for that ion. However, because the E239Q mutation could not be rescued, we propose that E239 forms a catalytic dyad with the bicarbonate to perform the proton abstraction necessary in the chemical pathway to the cyclopropane ring.

Flexibility in substrate recognition by thimet oligopeptidase as revealed by denaturation studies

Thimet oligopeptidase (TOP) is a soluble metalloendopeptidase belonging to a family of enzymes including neurolysin and neprilysin that utilize the HEXXH metal-binding motif. TOP is widely distributed among cell types and is able to cleave a number of structurally unrelated peptides. A recent focus of interest has been on structure-function relationships in substrate selectivity by TOP. The enzyme's structural fold comprises two domains that are linked at the bottom of a deep substrate-binding cleft via several flexible loop structures. In the present study, fluorescence spectroscopy has been used to probe structural changes in TOP induced by the chemical denaturant urea. Fluorescence emission, anisotropy and collisional quenching data support a two-step unfolding process for the enzyme in which complete loss of the tertiary structure occurs in the second step. Complete loss of activity and loss of catalytic Zn(II) from the active site, monitored by absorption changes of the metal chelator 4-(2-pyridylazo)-resorcinol, are also connected with the second step. In contrast, the first unfolding event, which is linked to changes in the non-catalytic domain, leads to a sharp increase in kcat towards a 9-residue substrate and a sharp decrease in kcat for a 5-residue substrate. Thus a conformational change in TOP has been directly correlated with a change in substrate selectivity. These results provide insight into how the enzyme can process the range of structurally unrelated peptides necessary for its many physiological roles.

The structure of cyclin E1/CDK2: implications for CDK2 activation and CDK2-independent roles

Cyclin E, an activator of phospho-CDK2 (pCDK2), is important for cell cycle progression in metazoans and is frequently overexpressed in cancer cells. It is essential for entry to the cell cycle from G0 quiescent phase, for the assembly of prereplication complexes and for endoreduplication in megakaryotes and giant trophoblast cells. We report the crystal structure of pCDK2 in complex with a truncated cyclin E1 (residues 81-363) at 2.25 A resolution. The N-terminal cyclin box fold of cyclin E1 is similar to that of cyclin A and promotes identical changes in pCDK2 that lead to kinase activation. The C-terminal cyclin box fold shows significant differences from cyclin A. It makes additional interactions with pCDK2, especially in the region of the activation segment, and contributes to CDK2-independent binding sites of cyclin E. Kinetic analysis with model peptide substrates show a 1.6-fold increase in kcat for pCDK2/cyclin E1 (81-363) over kcat of pCDK2/cyclin E (full length) and pCDK2/cyclin A. The structural and kinetic results indicate no inherent substrate discrimination between pCDK2/cyclin E and pCDK2/cyclin A with model substrates.

Protease Domain Sodium, Calcium, and Zinc Sites in Factor VIIa: Crystal Structures and Kinetic Studies.

During physiologic coagulation, the factor VIIa (FVIIa)/tissue factor (TF) complex activates FIX and FX. FVIIa consists of a N-terminal γ-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like (EGF) domains, and a C-terminal serine protease domain. We obtained crystals of FVIIa/soluble TF in the presence of Na+, Rb+, or Choline+ (Ch+) under conditions containing micromolar concentrations of Zn2+. Rb+ is a large monovalent ion and has been used to identify Na+-sites in several proteins; whereas, Ch+ cannot substitute for Na+. The various crystals diffracted from 2.0 to 2.4 Å and belonged to the space group P212121. In the crystal structures, Na+ or Rb+ in FVIIa coordinates to the carbonyl groups of residues 185 (chymotrypsin numbering), 185A, 221, and 224 as well as to two water molecules. Thus, the Na+-site in FVIIa is similar to that of FXa and activated protein C but not to that of thrombin. Ca2+ in the protease domain of FVIIa is coordinated to the carboxylates of Glu70 and Glu80 as seen earlier by Banner and coworkers. Additionally, the crystal structures also showed two Zn2+-sites, one involving His71 and the other involving His117. The Zn2+-sites are unique to FVIIa since the His residues are not present in other proteases. To investigate the role of Na+, Ca2+, and Zn2+-sites in the protease domain of FVIIa, a series of biochemical and kinetic studies were performed. Na+ increased the kcat for hydrolysis of S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide) ~22-fold by FVIIaWT whereas Ca2+ increased it ~by 230-fold. In the presence of Ca2+, Na+ had virtually no effect on the hydrolysis of S-2288; however, in the presence of Na+, Ca2+ increased the kcat ~12-fold. Thus, the increase in kcat by Ca2+ in the presence or absence of Na+ was similar (~250-fold). Further, Na+ had no effect on Km whereas Ca2+ increased it ~3.5-fold. However, the increase by in Km is biologically not pertinent since the Gla and EGF1 domains of FVIIa determine the Km for activation of FIX and FX. Moreover, FVIIaF225P (Na+-site mutant) showed little response to Na+ and FVIIaE80V (Ca2+-site mutant) showed no response to Ca2+ in hydrolyzing S-2288. These data indicate that the Na+ and Ca2+ effects observed are due to the occupancy of the protease domain Na+ and Ca2+ sites. Consistent with the Km data, Na+ had no effect on the binding of p-aminobenzamidine (pAB, S1 site probe) to FVIIaWT. Interestingly, Ca2+ decreased the Ki for pAB binding by ~5-fold indicating that the increase in Km for S-2288 caused by Ca2+ is not related to the S1 site but rather to the S2 and/or S3/S4 sites in FVIIa. In further studies, Zn2+ inhibited the potentiation of S-2288 hydrolysis by FVIIaWT with Ki ~1 of μM in the absence and ~30 μM in the presence of Ca2+. We conclude that the Na+-site in FVIIa is not linked to the synthetic substrate binding site(s), and that the Ca2+-site is linked to the substrate binding site(s). These observations are in contrast to what has been previously observed for FXa and activated protein C. Thus, in the absence of TF, Na+ and Ca2+ are positive regulators for catalysis by FVIIa; whereas, Zn2+ exerts a negative effect. Conceivably, occupancy of the Na+-site and the protease domain Ca2+-site may render FVIIa in a conformation suitable for TF binding and substrate hydrolysis. The local Zn2+ concentration following release by activated platelets at the site of hemostasis could dampen coagulation as a regulatory mechanism.

Aquifex aeolicus Aspartate Transcarbamoylase, an Enzyme Specialized for the Efficient Utilization of Unstable Carbamoyl Phosphate at Elevated Temperature

Aquifex aeolicus, an organism that flourishes at 95 degrees C, is one of the most thermophilic eubacteria thus far described. The A. aeolicus pyrB gene encoding aspartate transcarbamoylase (ATCase) was cloned, overexpressed in Escherichia coli, and purified by affinity chromatography to a homogeneous form that could be crystallized. Chemical cross-linking and size exclusion chromatography showed that the protein was a homotrimer of 34-kDa catalytic chains. The activity of A. aeolicus ATCase increased dramatically with increasing temperature due to an increase in kcat with little change in the Km for the substrates, carbamoyl phosphate and aspartate. The Km for both substrates was 30-40-fold lower than the corresponding values for the homologous E. coli ATCase catalytic subunit. Although rapidly degraded at high temperature, the carbamoyl phosphate generated in situ by A. aeolicus carbamoyl phosphate synthetase (CPSase) was channeled to ATCase. The transient time for carbamoyl aspartate formation was 26 s, compared with the much longer transient times observed when A. aeolicus CPSase was coupled to E. coli ATCase. Several other approaches provided strong evidence for channeling and transient complex formation between A. aeolicus ATCase and CPSase. The high affinity for substrates combined with channeling ensures the efficient transfer of carbamoyl phosphate from the active site of CPSase to that of ATCase, thus preserving it from degradation and preventing the formation of toxic cyanate.

Enhancement of enzymatic catalysis of Bacillus amyloliquefaciens α‐amylase by nonionic surfactant micelles

AbstractSome enzymes are considerably more stable when formulated with nonionic surfactants than when formulated with anionic surfactants. The effect of a nonionic surfatant, polyoxyethylene mono‐N‐dodecyl ether (Brij 35; number of units of ethylene oxide moieties, 23), on the kinetic behavior of hydrolysis of amylopectin with Bacillus amyloliquefaciens α‐amylase (BAA) was studied at a temperature of 25°C and a pH of 7.0. The hydrolytic rate was accelerated by the addition of the nonionic surfactant above its critical micelle concentration. Lineweaver‐Burk plots for the enzymatic hydrolysis in the absence and presence of the nonionic surfactant at 0.5 to 2.5% (wt/vol) had linear relationships, and the kinetic parameters, Km and kcat were obtained. The value of kcat was increased with an increased concentration of Brij 35, whereas the Km value was approximately constant. Therefore, the increase in kcat contributed to the acceleration of the apparent hydrolytic rate. The interaction of amylopectin with the surfactant was examined by a surface tension measurement, and the result confirmed the corresponding binding between the substrate and the surfactant. A fluorescence analysis due to tryptophan in BAA suggested that BAA bound to the nonionic micelles. The increase in kcat suggested that hydrolytic catalysis at the micellar pseudophase was more efficient than that at the aqueous pseudophase. The enhancement of the catalytic rate contributed to the effective removal of food stains containing starch when BAA was added with Brij 35 in a laundry detergent washing test.

Structural and Biochemical Evidence for an Autoinhibitory Role for Tyrosine 984 in the Juxtamembrane Region of the Insulin Receptor

Tyrosine 984 in the juxtamembrane region of the insulin receptor, between the transmembrane helix and the cytoplasmic tyrosine kinase domain, is conserved among all insulin receptor-like proteins from hydra to humans. Crystallographic studies of the tyrosine kinase domain and proximal juxtamembrane region reveal that Tyr-984 interacts with several other conserved residues in the N-terminal lobe of the kinase domain, stabilizing a catalytically nonproductive position of alpha-helix C. Steady-state kinetics measurements on the soluble kinase domain demonstrate that replacement of Tyr-984 with phenylalanine results in a 4-fold increase in kcat in the unphosphorylated (basal state) enzyme. Moreover, mutation of Tyr-984 in the full-length insulin receptor results in significantly elevated receptor phosphorylation levels in cells, both in the absence of insulin and following insulin stimulation. These data demonstrate that Tyr-984 plays an important structural role in maintaining the quiescent, basal state of the insulin receptor. In addition, the structural studies suggest a possible target site for small molecule activators of the insulin receptor, with potential use in the treatment of noninsulin-dependent diabetes mellitus.

Steady-state kinetics of MgATP splitting by native myosin RLC-free subfragment 1

MgATP positively regulates the dimerisation reaction, resulting in an increase in the rate of MgATP splitting with increasing MgATP concentration. We investigated the stoichiometry of this dimerisation reaction and found that each subunit in the dimer bound one molecule of MgATP at the dimerisation site. We studied changes with temperature in the MgATPase activity of S1 in the dimeric form for temperatures of 18–25°C. Between 18.0 and 21.2°C, kcat increased steadily with temperature. Between 21.2 and 21.8°C, there was a large decrease in kcat. A strong increase in kcat occurred at temperatures above 21.8°C, corresponding to a new reversible conformation of S1, unable to dimerise. The steep decrease in kcat between 21.2 and 21.8°C is due to a temperature transition in the monomer–dimer equilibrium.

Mechanism of hydrogen peroxide dismutation by a dimanganese catalase mimic: dominant role of an intramolecular base on substrate binding affinity and rate acceleration.

Several modifications of the manganese coordination environment and oxidation states of a family of synthetic dimanganese complexes have been introduced in search of the structural features that promote high rates of hydrogen peroxide dismutation (catalase activity). The X-ray structure of reduced catalase (T thermophilus) reveals a dimanganese(II,II) site linked by three bridges: mu 13-glutamate-, mu-OH-, and mu-OH2. The roles of a bridging hydroxide vs mu-aqua and the carboxylate have been examined in the reduced Mn2(II,II) complexes, [(L1,2)Mn2(mu-O2CCH3)(mu-X)]2+ for X- = OH- (7A) or X = H2O (1-4), and their oxidized Mn2(III,III) analogues, [(L1,2)Mn2(mu-O)(O2CCH3)(OH)]+ (6) (L1 is N,N,N',N'-tetrakis(2-methylenebenzamidazolyl)-1,3-diaminopropan- 2-ol, and L2 is the tetrakis-N-ethylated analogue of L1, which has all amine protons replaced by ethyl groups). The steady-state catalase rate is first-order in concentration of both substrate and reduced catalyst and saturates at high peroxide concentrations in all cases, confirming peroxide/catalyst complex formation. No catalyst decomposition is seen after > 2000 turnovers. Catalysis proceeds via a ping-pong mechanism between the Mn2(II,II/III,III) redox states, involving complexes 6 and 7A/7A'. The Mn2(III,IV) oxidation state was not active in catalase activity. Replacement of the mu-aqua bridge by mu-hydroxide eliminates a kinetic lag phase in production of the O2 product, increases the affinity for substrate peroxide in the rate-limiting step as seen by a 5-fold. decrease in the Michaelis constant (KM), and accelerates the maximum rate (kcat) by 65-fold The kinetic and spectroscopic data are consistent with substrate deprotonation by the hydroxide bridge, yielding a hydroperoxyl bridge coordinated between the Mn ions (mu, eta 2 geometry, "end-on") as the basis for catalysis: mu-OH- + H2O2-->mu-O2H- + H2O. Binding of a second hydroxide ion to 7A causes a further increase in kcat by 4-fold with no further change in substrate affinity (KM). By contrast, free (noncoordinating) bases in solution have no effect on catalysis, thus establishing intramolecular sites for both functional hydroxide anions. Solution structural studies indicate that the presence of 2-5 equiv of hydroxide in solution leads to formation of a bishydroxide species, [(L1,2)Mn2(mu 13-O2CCH3)(OH)2], which in the presence of air or oxygen auto-oxidizes to yield complex 6, a Mn2(III,III)(mu-O) species. Complex 6 oxidizes H2O2 to O2 without a kinetic lag phase and is implicated as the active form of the oxidized catalyst. A maximum increase by 240-fold in catalytic efficiency (kcat/KM = 700 s-1 M-1) is observed with the bishydroxide species versus the aquo complex 1, or only 800-fold less efficient than the enzyme. Deprotonation of the amine groups of the chelate ligand L was shown not to be involved in the hydroxide effects because identical results were obtained using the catalyst with tetrakis(N-ethylated)-L. Uncoupling of the Mn(II) spins by protonation of the alkoxyl bridge (LH) was observed to lower the catalase activity. Comparisons to other dimanganese complexes reveals that the Mn2(II,II)/Mn2(III,III) redox potential is not the determining factor in the catalase rate of these complexes. Rather, rate acceleration correlates with the availability of an intramolecular hydroxide for substrate deprotonation and with binding of the substrate at the bridging site between Mn ions in the reductive O-O bond cleavage step that forms water and complex 6.