Kinetic comparison of procaspase-3 and caspase-3

Caspases, the key enzymes in apoptosis, are synthesized as proenzymes and converted into active form by proteolytic cleavage. The residues on active site reorganize during the activation process as shown in the comparative studies of crystallographic structures of procaspase-7 and its mature form. On the other hand, the proenzyme itself has some activity. Aiming to characterize the activation process, the comparative kinetic study for the pro- and mature caspase-3 was performed. In 1/K(M) versus pH study, a residue with pKa of 6.89+/-0.13 was detected only in caspase-3. While Vmax versus pH kinetic results were consistent with the existence of a residue with pKa of 6.21+/-0.06 in procaspase-3 mutant (D9A/D28A/D175A) but not in caspase-3. In the inactivation assays with diethylpyrocarbonate, a residue (pKa, 6.61+/-0.05) could be determined only for caspase-3 whereas with iodoacetamide a residue with pKa value (6.01+/-0.05) could be assigned only for procaspase-3. Considering that those residues could be protected by caspase-3-specific inhibitor from the inactivation, the modifiers are histidine- and cysteine-specific, respectively, and the involvement of these residues in the characteristic catalytic dyad of caspases, the results indicate that the pKa values of the catalytic histidine and cysteine residues are changed during the activation process.     

Kinetic characterization of the protein Z-dependent protease inhibitor reaction with blood coagulation factor Xa

Protein Z-dependent protease inhibitor (ZPI) is a recently identified member of the serpin superfamily that functions as a cofactor-dependent regulator of blood coagulation factors Xa (FXa) and XIa. Here we show that ZPI and its cofactor, protein Z (PZ), inhibit procoagulant membrane-bound factor Xa by the branched pathway acyl-intermediate trapping mechanism used by other serpins, but with significant variations of this mechanism that are unique to ZPI. Rapid kinetic analyses showed that the reaction proceeded by the initial assembly of a membrane-associated PZ-ZPI-FXa Michaelis complex (K(M) 53+/-5 nM) followed by conversion to a stable ZPI-FXa complex (k(lim) 1.2+/-0.1 s(-1)). Cofactor premixing experiments together with independent kinetic analyses of ZPI-PZ and factor Xa-PZ-membrane complex formation suggested that assembly of the Michaelis complex through either ZPI-PZ-lipid or factor Xa-PZ-lipid intermediates was rate-limiting. Reaction stoichiometry analyses and native PAGE showed that for every factor Xa molecule inhibited by ZPI, two serpin molecules were cleaved. Native PAGE and immunoblotting showed that PZ dissociated from ZPI once ZPI forms a stable complex with FXa, and kinetic analyses confirmed that PZ acted catalytically to accelerate the membrane-dependent ZPI-factor Xa reaction. The ZPI-FXa complex was only transiently stable and dissociated with a rate constant that showed a bell-shaped pH dependence indicative of participation of factor Xa active-site residues. The complex was detectable by SDS-PAGE when denatured at low pH, consistent with it being a kinetically trapped covalent acyl-intermediate. Together our findings show that ZPI functions like other serpins to regulate the activity of FXa but in a manner uniquely dependent on protein Z, procoagulant membranes, and pH.     

Role of the reactive cysteine residue in restriction endonuclease Cfr9I.

Chemical modification studies were performed to elucidate the role of Cys-residues in the catalysis/binding of restriction endonuclease Cfr9I. Incubation of restriction endonuclease Cfr9I with N-ethylmaleimide (NEM), iodoacetate, 5,5'-dithiobis (2-nitrobenzoic acid) at pH 7.5 led to a complete loss of the catalytic activity. However, no enzyme inactivation was detectable after modification of the enzyme with iodoacetamide and methyl methanethiosulfonate. Complete protection of the enzyme against inactivation by NEM was observed in the presence of substrate implying that Cys-residues may be located at or in the vicinity of the active site of enzyme. Direct substrate-binding studies of native and modified restriction endonuclease Cfr9I using a gel-mobility shift assay indicated that the modification of the enzyme by NEM was hindered by substrate binding. A single Cys-residue was modified during the titration of the enzyme with DTNB with concomitant loss of the catalytic activity. The pH-dependence of inactivation of Cfr9I by NEM revealed the modification of the residue with the pKa value of 8.9 +/- 0.2. The dependence of the reaction rate of substrate hydrolysis by Cfr9I versus pH revealed two essential residues with pKa values of 6.3 +/- 0.15 and 8.7 +/- 0.15, respectively. The evidence presented suggests that the restriction endonuclease Cfr9I contains a reactive sulfhydryl residue which is non-essential for catalysis, but is located at or near the substrate binding site.     

Co-crystal structure and inhibition of factor Xa by PD0313052 identifies structurally stabilized active site residues of factor Xa and prothrombinase

The enzyme complex prothrombinase plays a pivotal role in fibrin clot development through the production of thrombin, making this enzyme complex an attractive target for therapeutic regulation. This study both functionally and structurally characterizes a potent, highly selective, active site directed inhibitor of human factor Xa and prothrombinase, PD0313052, and identifies structurally conserved residues in factor Xa and prothrombinase. Analyses of the association and dissociation of PD0313052 with human factor Xa identified a reversible, slow-onset mechanism of inhibition and a simple, single-step bimolecular association between factor Xa and PD0313052. This interaction was governed by association (k(on)) and dissociation (k(off)) rate constants of (1.0 +/- 0.1) x 10(7) M(-1) s(-1) and (1.9 +/- 0.5) x 10(-3) s(-1), respectively. The inhibition of human factor Xa by PD0313052 displayed significant tight-binding character described by a Ki* = 0.29 +/- 0.08 nM. Similar analyses of the inhibition of human prothrombinase by PD0313052 also identified a slow-onset mechanism with a Ki* = 0.17 +/- 0.03 nM and a k(on) and k(off) of (0.7 +/- 0.1) x 10(7) M(-1) s(-1) and (1.7 +/- 0.8) x 10(-3) s(-1), respectively. Crystals of factor Xa and PD0313052 demonstrated hydrogen bonding contacts within the S1-S4 pocket at residues Ser195, Asp189, Gly219, and Gly216, as well as interactions with aromatic residues within the S4 pocket. Overall, these data demonstrate that the inhibition of human factor Xa by PD0313052 occurs via a slow, tight-binding mechanism and indicate that active site residues of human factor Xa, including the catalytic Ser195, are effectively unaltered following assembly into prothrombinase.     

Inhibition of serine proteinases by tetra-p-amidinophenoxy-neo-pentane: thermodynamic and molecular modeling study

The inhibitory effect of the aromatic tetra-benzamidine derivative tetra-p-amidinophenoxy-neo-pentane (TAPP) on the catalytic properties of beta-trypsin (EC 3.4.21.4), alpha-thrombin (EC 3.4.21.5), factor Xa (EC 3.4.21.6), Lys77-plasmin (EC 3.4.21.7) and beta-kallikrein-B (EC 3.4.21.35) was investigated (between pH 2 and 8, I = 0.1 M; T = 37 +/- 0.5 degrees C), and analyzed in parallel with that of benzamidine, commonly taken as a molecular inhibitor model of serine proteinases. Over the whole pH range explored, TAPP and benzamidine show the same values of the dissociation inhibition constant (Ki) for beta-trypsin; at variance with the affinity of TAPP for alpha-thrombin, factor Xa, Lys77-plasmin and beta-kallikrein-B which is higher than that found for benzamidine association around neutrality, but tends to converge in the acidic pH limb. On lowering the pH from 5.5 to 3.0, values of Ki for TAPP binding to beta-trypsin as well as for benzamidine association to all the enzymes investigated decreased thus reflecting the pK-shift, upon inhibitor binding, of a single ionizing group. Over the same pH range, values of Ki for TAPP binding to alpha-thrombin, factor Xa, Lys77-plasmin and beta-kallikrein-B may be described as depending on the pK-shift, upon inhibitor association, of two equivalent proton-binding amino acid residues. Considering the X-ray three-dimensional structures and the computer-generated molecular models of serine proteinases: TAPP and :benzamidine adducts, the observed binding behaviour of TAPP and benzamidine to the enzymes considered has been related to the inferred stereochemistry of proteinase: inhibitor contact region(s).     

Reactivity of the essential thiol of Klebsiella aerogenes urease. Effect of pH and ligands on thiol modification

The kinetics of Klebsiella aerogenes urease inactivation by disulfide and alkylating agents was examined and found to follow pseudo-first-order kinetics. Reactivity of the essential thiol is affected by the presence of substrate and competitive inhibitors, consistent with a cysteine located proximal to the active site. In contrast to the results observed with other reagents, the rate of activity loss in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) saturated at high reagent concentrations, indicating that DTNB must first bind to urease before inactivation can occur. The pH dependence for the rate of urease inactivation by both disulfide and alkylating agents was consistent with an interaction between the thiol and a second ionizing group. The resulting macroscopic pKa values for the 2 residues are less than 5 and 12. Spectrophotometric studies at pH 7.75 demonstrated that 2,2'-dithiodipyridine (DTDP) modified 8.5 +/- 0.2 mol of thiol/mol of enzyme or 4.2 mol of thiol/mol of catalytic unit. With the slow tight binding competitive inhibitor phenyl-phosphorodiamidate (PPD) bound to urease, 1.1 +/- 0.1 mol of thiol/mol of catalytic unit were protected from modification. PPD-bound DTDP-modified urease could be reactivated by dialysis, consistent with the presence of one thiol per active site. Analogous studies at pH 6.1, using the competitive inhibitor phosphate, confirmed the presence of one protected thiol per catalytic unit. Under denaturing conditions, 25.5 +/- 0.3 mol of thiol/mol of enzyme (Mr = 211, 800) were modified by DTDP.     

The anomalous pKa of Tyr-9 in glutathione S-transferase A1-1 catalyzes product release

The pKa of the catalytic Tyr-9 in glutathione S-transferase (GST) A1-1 is lowered from 10.3 to approximately 8.1 in the apoenzyme and approximately 9.0 with a GSH conjugate bound at the active site. However, a clear functional role for the unusual Tyr-9 pKa has not been elucidated. GSTA1-1 also includes a dynamic C terminus that undergoes a ligand-dependent disorder-to-order transition. Previous studies suggest a functional link between Tyr-9 ionization and C-terminal dynamics. Here we directly probe the role of Tyr-9 ionization in ligand binding and C-terminal conformation. An engineered mutant of rGSTA1-1, W21F/F222W, which contains a single Trp at the C terminus, was used as a fluorescent reporter of pH-dependent C-terminal dynamics. This mutant exhibited a pH-dependent change in Trp-222 emission properties consistent with changes in C-terminal solvation or conformation. The apparent pKa values for the conformational transition were 7.9 +/- 0.1 and 9.3 +/- 0.1 for the apoenzyme and ligand-bound enzyme, respectively, in excellent agreement with the pKa for Tyr-9 in these states. The Y9F/W21F/F222W mutant, however, exhibited no such pH-dependent changes. Time-resolved fluorescence anisotropy studies revealed a ligand-dependent, Tyr-9-dependent, change in the order parameter of Trp-222. However, no pH dependence was observed. In equilibrium and pre-steady-state ligand binding studies, product conjugate had a decreased equilibrium binding affinity (KD), concomitant with increased binding and dissociation rates, at higher pH values. Furthermore, the recovered pKa values for the pH-dependent microscopic rate constants ranged from 7.7 to 8.4, also in agreement with the pKa of Tyr-9. In contrast, the Y9F/W21F/F222W mutant had no pH-dependent transition in KD or rate constants for ligand binding or dissociation. The combined results indicate that the macroscopic populations of "open" and "closed" states of the C terminus are not determined solely by the ionization state of Tyr-9. However, the rates of transition between these states are faster for the ionized Tyr-9. The ionized Tyr-9 states provide a parallel pathway for product dissociation, which is kinetically and thermodynamically favored. In silico kinetic models further support the functional role for the parallel dissociation pathway provided by ionized Tyr-9.     

pH dependence of the reaction rate of His 48 with p-bromophenacyl bromide and of the binding constant to Ca2+ of the monomeric forms of intact and alpha-NH2 modified phospholipases A2 from Trimeresurus flavoviridis

The phospholipase A2 of Trimeresurus flavoviridis was found to show monomer-dimer equilibria. Under conditions where the enzyme exists predominantly in the monomeric form, the chemical reaction rate of p-bromophenacyl bromide (BPB) with the catalytic group, His 48, was studied at 25 degrees C and ionic strength 0.2 by measuring the residual enzymic activity using a fluorescent substrate, 1,2-bis[4-(1-pyreno)butanoyl]-sn-glycero-3-phosphorylcholine (diPBPC). The pH-dependence curve of the reaction rate for the intact enzyme was practically the same as that for the modified enzyme, in which the N-terminal alpha-NH2 group had been selectively converted into an alpha-keto group. The pH-dependence curves were monophasic (sigmoidal) with a midpoint at pH 7.53, which corresponds to the pKa value of His 48. The pH dependences of the binding constants of Ca2+ to the intact and the alpha-NH2 modified enzymes were also studied at 25 degrees C and ionic strength 0.2 by measuring the changes in the tryptophyl fluorescence and/or aromatic CD spectra. The pH-dependence data for the modified enzyme were interpreted in terms of participation of Asp 49 (pKa 5.40) and His 48 (pKa 7.53), assuming that the protonation of Asp 49 competes with the Ca2+ binding. The pH-dependence data for the intact enzyme were similarly interpreted in terms of participation of the alpha-NH2 group (pKa 9.40) in addition to that of Asp 49 (pKa 5.40) and His 48 (pKa 7.53).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a functional role for histidine in lysyl oxidase catalysis

The pH-dependent kinetics of lysyl oxidase catalysis was examined for evidence of an ionizable enzyme residue which might function as a general base catalyzing proton abstraction previously shown to be a component of the mechanism of substrate processing by this enzyme. Plots of log Vmax/Km for the oxidation of n-hexylamine versus pH yielded pKa values of 7.0 +/- 0.1 and 10.4 +/- 0.1. The higher pKa varied with different substrates, reflecting ionization of the substrate amino group. A van't Hoff plot of the temperature dependence of the lower pKa yielded a value of 6.1 kcal mol-1 for the enthalpy of ionization. This value as well as the pKa of 7.0 are consistent with those of histidine residues previously implicated as general base catalysts in enzymes. Incubation of lysyl oxidase with low concentrations of diethyl pyrocarbonate, a histidine-selective reagent, at 22 degrees C and pH 7.0 irreversibly inhibited enzyme activity by a pseudo first-order kinetic process. The inactivation of lysyl oxidase correlated with spectral and pH-dependent kinetic evidence for the chemical modification of 1 histidine residue/mol of enzyme, the pKa of which was 6.9 +/- 0.1, within experimental error of that seen in the plot of log Vmax/Km versus pH. Enzyme activity was restored by incubation of the modified enzyme with hydroxylamine, consistent with the ability of this nucleophile to displace the carbethoxy group from N-carbethoxyhistidine. The presence of the n-hexylamine substrate largely protected against enzyme inactivation by diethyl pyrocarbonate. These results thus indicate a functional role for histidine in lysyl oxidase catalysis consistent with that of a general base in proton abstraction.     

Chemical and kinetic evidence for an essential histidine in the phosphotriesterase from Pseudomonas diminuta

The pH rate profile for the hydrolysis of diethyl-p-nitrophenyl phosphate catalyzed by the phosphotriesterase from Pseudomonas diminuta shows a requirement for the deprotonation of an ionizable group for full catalytic activity. This functional group has an apparent pKa of 6.1 +/- 0.1 at 25 degrees C, delta Hion of 7.9 kcal/mol, and delta Sion of -1.4 cal/K.mol. The enzyme is not inactivated in the presence of the chemical modification reagents dithiobis-(2-nitrobenzoate), methyl methane thiosulfonate, carbodiimide, pyridoxal, butanedione, or iodoacetic acid and thus cysteine, asparate, glutamate, lysine, and arginine do not appear to be critical for catalytic activity. However, the phosphotriesterase is inactivated completely with methylene blue, Rose Bengal, or diethyl pyrocarbonate. The enzyme is not inactivated by diethyl pyrocarbonate in the presence of bound substrate analogs, and inactivation with diethyl pyrocarbonate is reversible upon addition of neutralized hydroxylamine. The modification of a single histidine residue by diethyl pyrocarbonate, as shown by spectrophotometric analysis, is responsible for the loss of catalytic activity. The pKinact for diethyl pyrocarbonate modification is 6.1 +/- 0.1 at 25 degrees C. These results have been interpreted to suggest that a histidine residue at the active site of phosphotriesterase is facilitating the reaction by general base catalysis.     

4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole as a reactivity probe for the investigation of the thiol proteinases. evidence that ficin and bromelain may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain.

1. 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride) was used as a reactivity probe to characterize the active centres of papin (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). 2. In the pH range 0-8 Nbd chloride probably exists mainly as a monocation, possibly with the proton located on N-1 of the oxadiazole ring. 3. Spectroscopic evidence is presented for the intermediacy of Meisenheimer-type adducts in the reaction of Nbd chloride with nucleophiles. 4. The pH-dependence of the second-order rate constants (k) of the reactions of the three enzymes with Nbd chloride was determined at 25 degrees C, I = 0.1 mol/litre in 6.7% (v/v) ethanol in the pH range 2.5-5, where, at least for papain and ficin, the reactions occur specifically with their active-centre thiol groups. The pH-k profile for the papain reaction is bell-shaped (pKaI = 3.24, pKaII = 3.44 and k = 86M(-1)-s(-1), whereas that for ficin is sigmoidal (pKa = 3.6, k = 0.36M(-1)-s(-1), the rate increasing with increasing pH. The profile for the bromelain reaction appears to resemble that for the ficin reaction, but is complicated by amino-group labelling. 5. The bell-shaped profile of the papain reaction is considered to arise from the reaction of the thiolate ion of cysteine-25, maintained in acidic media by interaction with the side chain of histidine-159, with the Nbd chloride monocation hydrogen-bonded at its nitro group to the un-ionized form of the carboxyl group of aspartic acid-158. The lack of acid catalysis in the corresponding reactions of ficin and probably of bromelain suggests that these enzymes may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain. The possible consequences of this for the catalytic sites of these enzymes is discussed.     

Inactivation of human fibroblast collagenase by chloroacetyl N-hydroxypeptide derivatives.

When human fibroblast collagenase was incubated with ClCH2CO-(N-OH)Leu-Ala-Gly-NH2 (2-5 mM) in Tris buffer, pH 7.4 at 25 degrees C, a slow, time-dependent inhibition of the enzyme was observed. Dialysis against a buffer to remove free inhibitor did not reactivate the enzyme. A reversible competitive inhibitor, phthaloyl-GlyP-Ile-Trp-NHBzl (50 microM) partially protected the enzyme from inactivation by the compound. From the concentration dependent rates of inactivation Ki = 0.5 +/- 0.1 mM and k3, the rate constant for inactivation = 3.4 +/- 0.3 x 10(-3) min-1 were determined. The inactivation followed the pH optimum (6.5-7.0) for the enzyme activity, suggesting direct involvement of the same active site residue(s). The reaction mode of the inhibitor may be analogous to that of the inactivation of Pseudomonas aeruginosa elastase [Nishino, N. and Powers, J. (1980) J. Biol. Chem., 255, 3482] in which the catalytic glutamate carboxyl was alkylated by the inhibitor after its binding to enzyme through the hydroxamic Zn2+ ligand. All carboxyl groups in the inactivated collagenase were modified with 0.1 M ethyl dimethylaminopropyl carbodiimide/0.5 M glycinamide in 4 M guanidine at pH 5. The inactivator-affected carboxyl group was then regenerated with 1 M imidazole at pH 8.9, 37 degrees C for 12 h and the protein was radiolabeled with 3H-glycine methyl ester and carbodiimide to incorporate 0.9 residue glycine per mol enzyme.     

Mg2+ -dependent inactivation / H+ -dependent activation equilibrium of chloroplast F1-ATPase

The catalytic part of chloroplast thylakoid ATPase, the chloroplast coupling factor CF1, is reversibly inactivated during incubation in the presence of Mg2+. The inactivation has two phases. Its fast phase occurs at basic pH of the incubation medium (k = 6 min-1), while the slow phase ( k = 0.1-0.2 min-1) depends on pH only slightly throughout the studied range (5.5-9.0). As followed from changes in the inactivation effect of magnesium ions, Mg2+ affinity for the enzyme decreases dramatically with decreasing medium pH. The pH-dependence of Mg2+ dissociation apparent constant suggests that the binding/dissociation equilibrium is determined by protonation/deprotonation of specific acid-base groups of the enzyme. The analysis of pH-dependence plots gives the equilibrium constant of magnesium dissociation (3-9 M) and the dissociation constant of the protonated groups pK 5.8-6.7). Sodium azide is known to stabilize the inactive CF1-MgADP complex; when added to the incubation medium it diminishes the Mg2+ dissociation constant and has no effect on the dissociation constant of the acid-base groups. At lower pH, Mg2+-inactivated CF1-ATPase reactivates. Octyl glucoside accelerates the reactivation, while Triton-100 affects it only slightly. The reactivation rate of membrane-bound CF1 (thylakoid ATPase) inactivated by preincubation with Mg2+ in the presence of gramicidin is a few times higher than that of isolated CF1. These results suggest that the reactivation of isolated and membrane-bound CF1-ATPase is determined by protonation of a limited number of acid-base groups buried in the enzyme molecule.     

The enthalpy of protolysis of liver alcohol dehydrogenase upon binding nicotinamide adenine dinucleotide.

The binding of NAD+, NADH, and ADP-ribose to horse liver alcohol dehydrogenase has been studied calorimetrically as a function of pH at 25 degrees C. The enthalpy of NADH binding is 0 +/- 0.5 kcal mol-1 in the pH range 6 to 8.6. The enthalpy of NAD+ binding, however, varies with pH in a sigmoidal fashion and is -4.0 kcal mol(NAD)-1 at pH 6.0 and +4.5 kcal mol(NAD)-1 at pH 8.6 with an apparent pKa of 7.6 +/- 0.2. The enthalpy of proton ionization of the group on the enzyme is calculated to be in the range 8.8 to 9.8 kcal mol(H+)-1. In conjunction with the available thermodynamic data on the ionization of zinc-bound water in model compounds, it is concluded that the group with a pKa of 9.8 in the free enzyme and 7.6 in the enzyme . NAD+ binary complex is, most likely, the zinc-bound water molecule. Our studies with zinc-free enzyme provide further evidence for this conclusion. Therefore, the processes involving a conformational change of the enzyme upon NAD+ binding and the suggested mechanism of subsequent quenching of the fluorescence of Trp-314 implicating the participation of an ionized tyrosine group must be re-evaluated in the light of this thermodynamic study.     

Kinetics of inactivation of membrane-bound factor Va by activated protein C. Protein S modulates factor Xa protection

Kinetic analyses were done to determine what effect factor Xa and protein S had on the activated protein C (APC)-catalyzed inactivation of factor Va bound to phospholipid vesicles or human platelets. In the presence of optimal concentrations of phospholipid vesicles and Ca2+, a Km of 19.7 +/- 0.6 nM factor Va and a kcat of 23.7 +/- 10 mol of factor Va inactivated/mol of APC/min were obtained. Added purified plasma protein S increased the maximal rate of factor Va inactivation only 2-fold without effect on the Km. Protein S effect was unaltered when the phospholipid concentration was varied by 2 orders of magnitude. The reaction on unactivated human platelets yielded a Km = 12.5 +/- 2.6 nM and kcat = 6.2 +/- 0.6 mol of factor Va inactivated/mol of APC/min. Added purified plasma protein S or release of platelet protein S by platelet activation doubled the kcat value without affecting the Km. Addition of a neutralizing anti-protein S antibody abrogated the effect of plasma protein S or platelet-released protein S, but was without effect in the absence of plasma protein S or platelet activation. Studies with factor Xa indicated that factor Xa protects factor Va from APC-catalyzed inactivation by lowering the effective concentration of factor Va available to interact with APC. From these data a dissociation constant of less than 0.5 nM was calculated for the interaction of factor Xa with membrane-bound factor Va. Protein S abrogated the ability of factor Xa to protect factor Va from inactivation by APC without affecting the interaction of factor Xa with factor Va. These combined data suggest that one physiological function of protein S is to allow the APC-catalyzed inactivation of factor Va in the presence of factor Xa.     

Soybean nodule xanthine dehydrogenase: a kinetic study

Xanthine dehydrogenase was purified from soybean nodules and the kinetic properties were studied at pH 7.5. Km values of 5.0 +/- 0.6 and 12.5 +/- 2.5 microM were obtained for xanthine and NAD+, respectively. The pattern of substrate dependence suggested a Ping-Pong mechanism. Reaction with hypoxanthine gave Km's of 52 +/- 3 and 20 +/- 2.5 microM for hypoxanthine and NAD+, respectively. The Vmax for this reaction was twice that for the xanthine-dependent reaction. The pH dependence of Vmax gave a pKa of 7.6 +/- 0.1 for either xanthine or hypoxanthine oxidation. In addition the Km for xanthine had a pKa of 7.5 consistent with the protonated form of xanthine being the true substrate. Km for hypoxanthine varied only 2.5-fold between pH 6 and 10.7. Product inhibition studies were carried out with urate and NADH. Both products gave mixed inhibition with respect to both substrates. Xanthine dehydrogenase was able to use APAD+ as an electron acceptor for xanthine oxidation, with a Km at pH 7.5 of 21.2 +/- 2.5 microM and Vmax the same as that obtained with NAD+. Reduction of APAD+ by NADH was also catalyzed by xanthine dehydrogenase with a Km of 102 +/- 15 microM; Vmax was approximately 2.5 times that for the xanthine-dependent reaction, and was independent of pH between 6 and 9. Reaction with group-specific reagents indicated the possibility of an essential histidyl group. A thiol-modifying reagent did not cause inactivation of the enzyme. A role for the histidyl side chain in catalysis is proposed.     

Extraordinary rates of transition metal ion-mediated ribozyme catalysis

In pre-steady-state, fast-quench kinetic analysis, the tertiary-stabilized hammerhead ribozyme "RzB" cleaves its substrate RNA with maximal measured k (obs) values of approximately 3000 min(-1) in 1 mM Mn(2+) and approximately 780 min(-1) in 1 mM Mg(2+) at 37 degrees C (pH 7.4). Apparent pKa for the catalytic general base is approximately 7.8-8.5, independent of the corresponding metal hydrate pKa, suggesting potential involvement of a nucleobase as general base as suggested previously from nucleobase substitution studies. The pH-rate profile is bell-shaped for Cd(2+), for which the general catalytic acid has a pKa of 7.3 +/- 0.1. Simulations of the pH-rate relation suggest a pKa for the general catalytic acid to be approximately 9.5 in Mn(2+) and >9.5 in Mg(2+). The acid pKa's follow the trend in the pKa of the hydrated metal ions but are displaced by approximately 1-2 pH units in the presence of Cd(2+) and Mn(2+). One possible explanation for this trend is direct metal ion coordination with a nucleobase, which then acts as general acid.     

Standard free energy for the hydrolysis of adenylylated T4 DNA ligase and the apparent pKa of lysine 159

Equilibrium constants for the adenylylation of T4 DNA ligase have been measured at 10 pH values. The values, when plotted against pH, fit a titration curve corresponding to a pKa of 8.4 +/- 0.1. The simplest interpretation is that the apparent pKa is that of the 6-amino group of the AMP-accepting residue Lys159. Based on the pH dependence of the equilibrium constants, the value at pH 7.0 is 0. 0213 at 25 degrees C, corresponding to DeltaG'o = +2.3 kcal mol-1. From this value and the standard free energy change of -10.9 kcal mol-1 for the hydrolysis of ATP to AMP and PPi, we calculate that DeltaG'o for the hydrolysis of the adenylyl-DNA ligase is -13.2 kcal mol-1. The presence of conserved basic amino acid residues in the catalytic domain, which are proximal to the active site in the homologous catalytic domain of T7 DNA ligase, suggests that the pKa of Lys159 is perturbed downward by the electrostatic effects of nearby positively charged amino acid side chains. The lower than normal pKa 8.4 compared with 10.5 for the 6-amino group of lysine and the high energy of the alpha,beta-phosphoanhydride linkage in ATP significantly facilitate adenylylation of the enzyme.     

Modification of an arginine residue essential for the activity of NAD-malic enzyme from Ascaris suum

Purified NAD-malic enzyme from Ascaris suum is rapidly inactivated by the arginine reagent, 2,3-butanedione, and this inactivation is facilitated by 30 mM borate. Determination of the inactivation rate as a function of butanedione concentration suggests a second-order process overall, which is first order in butanedione. A second-order rate constant of 0.6 M-1 s-1 at pH 9 is obtained for the butanedione reaction. The inactivation is reversed by removal of the excess reagent upon dialysis. The enzyme is protected against inactivation by saturating amounts of malate in the presence and absence of borate. The divalent metal Mg2+ affords protection in the presence of borate but has no effect in its absence. The nucleotide reactant NAD+ has no effect on the inactivation rate in either the presence or absence of borate. A dissociation constant of 24 mM is obtained for E:malate from the decrease in the inactivation rate as a function of malate concentration. An apparent Ki of 0.5 mM is obtained for oxalate (an inhibitor competitive vs malate) from E:Mg:oxalate while no significant binding is observed for oxalate using the butanedione modified enzyme. The pH dependence of the first-order rate of inactivation by butanedione gives a pKa of 9.4 +/- 0.1 for the residue(s) modified, and this pK is increased when NAD is bound. The arginine(s) modified is implicated in the binding of malate.     

The influence of pH on the interaction of inhibitors with triosephosphate isomerase and determination of the pKa of the active-site carboxyl group.

Ionization effects on the binding of the potential transition state analogues 2-phosphoglycolate and 2-phosphoglycolohydroxamate appear to be attributable to the changing state of ionization of the ligands themselves, therefore it is unnecessary to postulate the additional involvement of an ionizing residue at the active site of triosephosphate isomerase to explain the influence of changing pH on Ki in the neutral range. The binding of the competitive inhibitor inorganic sulfate is insensitive to changing pH in the neutral range. 3-Chloroacetol sulfate, synthesized as an active-site-specific reagent for triosephosphate isomerase, is used to provide an indication of the pKa of the essential carboxyl group of this enzyme. Previously described active-site-specific reagents for the isomerase were phosphate esters, and their changing state of ionization (accompanied by possible changes in their affinity for the active site) may have complicated earlier attempts to determine the pKa of the essential carboxyl group from the pH dependence of the rate of inactivation. Being a strong monoprotic acid, chloroacetol sulfate is better suited to the determination of the pKa of the carboxyl group. Chloroacetol sulfate inactivates triosephosphate isomerase by the selective esterification of the same carboxyl group as that which is esterified by the phosphate esters described earlier. From the pH dependence of the rate of inactivation of yeast triosephosphate isomerase, the apparent pKa of the active-site carboxyl group is estimated as 3.9 +/- 0.1.