Kinetics of the hydrolysis of N-benzoyl-l-serine methyl ester catalysed by bromelain and by papain. Analysis of modifier mechanisms by lattice nomography, computational methods of parameter evaluation for substrate-activated catalyses and consequences of postulated non-productive binding in bromelain- and papain-catalysed hydrolyses

1. N-Benzoyl-l-serine methyl ester was synthesized and evaluated as a substrate for bromelain (EC 3.4.22.4) and for papain (EC 3.4.22.2). 2. For the bromelain-catalysed hydrolysis at pH7.0, plots of [S(0)]/v(i) (initial substrate concn./initial velocity) versus [S(0)] are markedly curved, concave downwards. 3. Analysis by lattice nomography of a modifier kinetic mechanism in which the modifier is substrate reveals that concave-down [S(0)]/v(i) versus [S(0)] plots can arise when the ratio of the rate constants that characterize the breakdown of the binary (ES) and ternary (SES) complexes is either less than or greater than 1. In the latter case, there are severe restrictions on the values that may be taken by the ratio of the dissociation constants of the productive and non-productive binary complexes. 4. Concave-down [S(0)]/v(i) versus [S(0)] plots cannot arise from compulsory substrate activation. 5. Computational methods, based on function minimization, for determination of the apparent parameters that characterize a non-compulsory substrate-activated catalysis are described. 6. In an attempt to interpret the catalysis by bromelain of the hydrolysis of N-benzoyl-l-serine methyl ester in terms of substrate activation, the general substrate-activation model was simplified to one in which only one binary ES complex (that which gives rise directly to products) can form. 7. In terms of this model, the bromelain-catalysed hydrolysis of N-benzoyl-l-serine methyl ester at pH7.0, I=0.1 and 25 degrees C is characterized by K(m) (1) (the dissociation constant of ES)=1.22+/-0.73mm, k (the rate constant for the breakdown of ES to E+products, P)=1.57x10(-2)+/-0.32x10(-2)s(-1), K(a) (2) (the dissociation constant that characterizes the breakdown of SES to ES and S)=0.38+/-0.06m, and k' (the rate constant for the breakdown of SES to E+P+S)=0.45+/-0.04s(-1). 8. These parameters are compared with those in the literature that characterize the bromelain-catalysed hydrolysis of alpha-N-benzoyl-l-arginine ethyl ester and of alpha-N-benzoyl-l-arginine amide; K(m) (1) and k for the serine ester hydrolysis are somewhat similar to K(m) and k(cat.) for the arginine amide hydrolysis and K(as) and k' for the serine ester hydrolysis are somewhat similar to K(m) and k(cat.) for the arginine ester hydrolysis. 9. A previous interpretation of the inter-relationships of the values of k(cat.) and K(m) for the bromelain-catalysed hydrolysis of the arginine ester and amide substrates is discussed critically and an alternative interpretation involving substantial non-productive binding of the arginine amide substrate to bromelain is suggested. 10. The parameters for the bromelain-catalysed hydrolysis of the serine ester substrate are tentatively interpreted in terms of non-productive binding in the binary complex and a decrease of this type of binding by ternary complex-formation. 11. The Michaelis parameters for the papain-catalysed hydrolysis of the serine ester substrate (K(m)=52+/-4mm, k(cat.)=2.80+/-0.1s(-1) at pH7.0, I=0.1, 25.0 degrees C) are similar to those for the papain-catalysed hydrolysis of methyl hippurate. 12. Urea and guanidine hydrochloride at concentrations of 1m have only small effects on the kinetic parameters for the hydrolysis of the serine ester substrate catalysed by bromelain and by papain.     

Enantioselective hydrolysis of naproxen ethyl ester catalyzed by monoclonal antibodies

This report described that a hapten of racemic phosphonate 3 designed as the mimic of the transition state of hydrolysis of naproxen ethyl ester was successfully synthesized from easily available 2-acetyl-6-methoxy-naphthalene 5. Then BALB/C mice were immunized and one of the monoclonal catalytic antibodies, N116-27, which enantioselectively accelerated the hydrolysis of the R-(-)-naproxen ethyl ester was given. The Michaelis-Menton parameter for the catalyzed reaction was K(M)=6.67 mM and k(cat)/k(uncat)=5.8 x 10(4). This enantioselective result was explained by the fact that the R-isomer of rac-hapten was more immunogenic than the S-isomer.     

Pre-steady-state and stopped-flow fluorescence analysis of Escherichia coli ribonuclease III: insights into mechanism and conformational changes associated with binding and catalysis

To better understand substrate recognition and catalysis by RNase III, we examined steady-state and pre-steady-state reaction kinetics, and changes in intrinsic enzyme fluorescence. The multiple turnover cleavage of a model RNA substrate shows a pre-steady-state burst of product formation followed by a slower phase, indicating that the steady-state reaction rate is not limited by substrate cleavage. RNase III catalyzed hydrolysis is slower at low pH, permitting the use of pre-steady-state kinetics to measure the dissociation constant for formation of the enzyme-substrate complex (K(d)=5.4(+/-0.6) nM), and the rate constant for phosphodiester bond cleavage (k(c)=1.160(+/-0.001) min(-1), pH 5.4). Isotope incorporation analysis shows that a single solvent oxygen atom is incorporated into the 5' phosphate of the RNA product, which demonstrates that the cleavage step is irreversible. Analysis of the pH dependence of the single turnover rate constant, k(c), fits best to a model for two or more titratable groups with pK(a) of ca 5.6, suggesting a role for conserved acidic residues in catalysis. Additionally, we find that k(c) is dependent on the pK(a) value of the hydrated divalent metal ion included in the reaction, providing evidence for participation of a metal ion hydroxide in catalysis, potentially in developing the nucleophile for the hydrolysis reaction. In order to assess whether conformational changes also contribute to the enzyme mechanism, we monitored intrinsic tryptophan fluorescence. During a single round of binding and cleavage by the enzyme we detect a biphasic change in fluorescence. The rate of the initial increase in fluorescence was dependent on substrate concentration yielding a second-order rate constant of 1.0(+/-0.1)x10(8) M(-1) s(-1), while the rate constant of the second phase was concentration independent (6.4(+/-0.8) s(-1); pH 7.3). These data, together with the unique dependence of each phase on divalent metal ion identity and pH, support the hypothesis that the two fluorescence transitions, which we attribute to conformational changes, correlate with substrate binding and catalysis.     

Escherichia coli cytosine deaminase: the kinetics and thermodynamics for binding of cytosine to the apoenzyme and the Zn(2+) holoenzyme are similar

Recombinant Escherichia coli cytosine deaminase is purified as a mixture of Zn(2+) and Fe(2+) forms of the enzyme. Fe(2+) is removed readily by o-phenanthroline to yield apoenzyme (apoCDase) that contains <0.2 mol of Zn(2+)per mol of subunit. ApoCDase was efficiently reconstituted to Zn(2+)CDase by treatment with ZnCl(2). The interaction of cytosine with apoCDase and Zn(2+)CDase was investigated at pH 7.5 and 25 degrees C by monitoring changes in intrinsic protein fluorescence. The values for the kinetic data K(1), k(2), and k(3) for Zn(2+)CDase were 0.25 mM, 80 s(-1), and 38 s(-1), respectively. The value for k(-2) was statistically indistinguishable from zero. The analogous values for K(1), k(2), and k(-2), (k(3)=0) for apoCDase were 0.157 mM, 186 s(-1) and approximately 0.8 s(-1), respectively. The overall dissociation constant of apoCDase for cytosine was 0.00069 mM, whereas the K(m) of Zn(2+)CDase for cytosine was 0.20 mM. The pre-steady state phase of the reaction was associated with an absorbance increase at 280 nm that was attributed to solvent perturbation of the spectrum of cytosine or enzyme. Formation of the Fe(2+)CDase-cytosine complex was too rapid to monitor by these techniques.     

Relationship of exo-beta-D-galactofuranosidase kinetic parameters to the number of phosphodiesters in Penicillium fellutanum peptidophosphogalactomannan: enzyme purification and kinetics of glycopeptide and galactofuran chain hydrolysis

Extracellular Penicillium fellutanum exo-beta-D-galactofuranosidase, with a mass of 70 kDa, was purified to apparent homogeneity. The enzyme was used to investigate the influence of phosphodiesters of the peptidophosphogalactomannans pP(2)GM(ii) and pP(25)GM(ii) (containing 2 and 25 phosphodiester residues, respectively, per mol of polymer) on the kinetic parameters of galactofuranosyl hydrolysis of these two polymers, of 1-O-methyl-beta-D-galactofuranoside, and of two galactofuranooligosaccharides. The enzyme did not hydrolyze phosphorylated galactose residues of pP(2)GM(ii) or pP(25)GM(ii). The k(cat)/K(m) value for pP(25)GM(ii) is 1.7 x 10(3) M(-1) s(-1), that for 1-O-methyl-beta-D-galactofuranoside is 1.1 x 10(4) M(-1) s(-1), that for pP(2)GM(ii) is 1.7 x 10 (4) M(-1) s(-1), and those for 5-O-beta-D-galactofuranooligosaccharides with degrees of polymerization of 3.4 and 5.5 are 1.7 x 10(5) and 4.1 x 10(5) M(-1) s(-1), respectively. Variability in the k(cat)/K(m) values is due primarily to differences in K(m) values; the k(-1)/k(1) ratio likely provides the most influence on K(m). k(cat) increases as the degree of polymerization of galactofuranosyl residues increases. Most of the galactofuranosyl and phosphocholine residues were removed by day 8 in vivo from pP(x)GM(ii) added to day 3 cultures initiated in medium containing 2 mM phosphate but not from those initially containing 20 mM phosphate. The filtrates from day 9 cultures initiated in 2 mM inorganic phosphate in modified Raulin-Thom medium contained 0.2 mM inorganic phosphate and 2.2 U of galactofuranosidase ml(-1)h(-1). No galactofuranosidase activity but 15 mM inorganic phosphate was found in filtrates from day 9 cultures initiated in 20 mM phosphate. In vivo the rate of galactofuranosyl hydrolysis of pP(x)GM(ii) and of related polymers is proportional to the k(cat)/K(m) value of each polymer. The kinetic data show that the k(cat)/K(m) value increases as the number of phosphodiesters of pP(x)GM(ii) decreases, also resulting in an increase in the activity of exo-beta-D-galactofuranosidase.     

Kinetic properties of human placental glucose-6-phosphate dehydrogenase

The kinetic properties of placental glucose-6-phosphate dehydrogenase were studied, since this enzyme is expected to be an important component of the placental protection system. In this capacity it is also very important for the health of the fetus. The placental enzyme obeyed "Rapid Equilibrium Ordered Bi Bi" sequential kinetics with K(m) values of 40+/-8 microM for glucose-6-phosphate and 20+/-10 microM for NADP. Glucose-6-phosphate, 2-deoxyglucose-6-phosphate and galactose-6-phosphate were used with catalytic efficiencies (k(cat)/K(m)) of 7.4 x 10(6), 4.89 x 10(4) and 1.57 x 10(4) M(-1).s(-1), respectively. The K(m)app values for galactose-6-phosphate and for 2-deoxyglucose-6-phosphate were 10+/-2 and 0.87+/-0.06 mM. With galactose-6-phosphate as substrate, the same K(m) value for NADP as glucose-6-phosphate was obtained and it was independent of galactose-6-phosphate concentration. On the other hand, when 2-deoxyglucose-6-phosphate used as substrate, the K(m) for NADP decreased from 30+/-6 to 10+/-2 microM as the substrate concentration was increased from 0.3 to 1.5 mM. Deamino-NADP, but not NAD, was a coenzyme for placental glucose-6-phosphate dehydrogenase. The catalytic efficiencies of NADP and deamino-NADP (glucose-6-phosphate as substrate) were 1.48 x 10(7) and 4.80 x 10(6) M(-1)s(-1), respectively. With both coenzymes, a hyperbolic saturation and an inhibition above 300 microM coenzyme concentration, was observed. Human placental glucose-6-phosphate dehydrogenase was inhibited competitively by 2,3-diphosphoglycerate (K(i)=15+/-3 mM) and NADPH (K(i)=17.1+/-3.2 microM). The small dissociation constant for the G6PD:NADPH complex pointed to tight enzyme:NADPH binding and the important role of NADPH in the regulation of the pentose phosphate pathway.     

Purification and characterization of the beta-trefoil fold protein barley alpha-amylase/subtilisin inhibitor overexpressed in Escherichia coli

Barley alpha-amylase/subtilisin inhibitor (BASI) is a beta-trefoil fold protein related to soybean trypsin inhibitor (Kunitz) and inhibits barley alpha-amylase isozyme 2 (AMY2), which is de novo synthesized in the seed during germination. Recombinant BASI was produced in Escherichia coli in an untagged form (untagged rBASI), in two His(6)-tag forms (His(6)-rBASI and His(6)-Xa-rBASI), and in an intein-CBD-tagged form (rBASI (intein)). The yields per liter culture after purification were (i) 25 mgl(-1) His(6)-rBASI; (ii) 6 mgl(-1) rBASI purified after cleavage of His(6)-Xa-rBASI by Factor Xa; (iii) 3 mgl(-1) untagged rBASI; and (iv) 0.2 mgl(-1) rBASI after a chitin-column and autohydrolysis of the rBASI-intein-CBD. In Pichia pastoris, rBASI was secreted at 0.1 mgl(-1). The recombinant BASI forms and natural seed BASI (sBASI) all had an identical isoelectric point of 7.2 and a mass of 19,879 Da, as determined by mass spectrometry. The fold of rBASI from the different preparations was confirmed by circular dichroism spectroscopy and rBASI (intein), His(6)-rBASI, and sBASI inhibited AMY2 catalyzed starch hydrolysis with K(i) of 0.10, 0.06, and 0.09 nM, respectively. Surface plasmon resonance analysis of the formation of AMY2/rBASI (intein) gave k(on)=1.3x10(5)M(-1)s(-1), k(off)=1.4x10(-4)s(-1), and K(D)=1.1 nM, and of the savinase-His(6)-rBASI complex k(on)=21.0x10(4)M(-1)s(-1), k(off)=53.0x10(-4)s(-1), and K(D)=25.0 nM, in agreement with sBASI values. K(i) was 77 and 65 nM for inhibition of savinase activity by His(6)-rBASI and sBASI, respectively.     

Mechanistic studies of ubiquitin C-terminal hydrolase L1

Ubiquitin C-terminal hydrolases (UCHs) cleave Ub-X bonds (Ub is ubiquitin and X an alcohol, an amine, or a protein) through a thioester intermediate that is produced by nucleophilic attack of the Cys residue of a Cys-SH/His-Im catalytic diad. We are studying the mechanism of UCH-L1, a UCH that is implicated in Parkinson's disease, and now wish to report our initial findings. (i) Pre-steady-state kinetic studies for UCH-L1-catalyzed hydrolysis of Ub-AMC (AMC, 7-amido-4-methylcoumarin) indicate that k(cat) is rate-limited by acyl-enzyme formation. Thus, K(m) = K(s), the dissociation constant for the Michaelis complex, and k(cat) = k(2), the rate constant for acyl-enzyme formation. (ii) For K(assoc) (=K(s)(-)(1)), DeltaC(p) = -0.8 kcal mol(-)(1) deg(-)(1) and is consistent with coupling between substrate association and a conformational change of the enzyme. For k(2), DeltaS(++) = 0 and suggests that in the E-S, substrate and active site residues are precisely aligned for reaction. (iii) Solvent isotope effects are (D)K(assoc) = 0.5 and (D)k(2) = 0.9, suggesting that the substrate binds to a form of free enzyme in which the active site Cys exists as the thiol. In the resultant Michaelis complex, the diad has tautomerized to ion pair Cys-S(-)/His-ImH(+). Subsequent attack of thiolate produces the acyl-enzyme species. In contrast, isotope effects for association of UCH-L1 with transition-state analogue ubiquitin aldehyde suggest that an alternative mechanistic pathway can sometimes be available to UCH-L1 involving general base-catalyzed attack of Cys-SH by His-Im.     

Kinetics of action of chymosin (rennin) on some peptide bonds of bovine beta-casein

The first steps of proteolysis of bovine beta-casein by chymosin were studied quantitatively by using reverse-phase high-performance liquid chromatography (RP-HPLC). Although chymosin has a broad specificity, it has been possible to selectively study the hydrolysis of two bonds (Ala-189-Phe-190 and Leu-192-Tyr-193) by choosing appropriate conditions. The disappearance of the substrate and the appearance of the reaction products as a function of time were followed at 220 nm by RP-HPLC. For concentrations where beta-casein was in a micellar form, the Michaelian parameters corresponding to the cleavage of bond 192-193 were determined by measuring initial rates of reaction at different substrate concentrations in a time period for which splitting of bond 189-190 was negligible. The following results were obtained; k1cat = 1.54 s-1, K1m = 0.075 mM, and k1cat/K1m = 20.6 mM-1 s-1. Under conditions where the protein was in a monomeric state, the following parameters were determined for the splitting of bond 192-193 by integrating the Michaelis equation: k2cat = 0.056 s-1, K2m = 0.007 mM, and k2cat/K2m = 79.7 mM-1 s-1. Under the latter conditions the four enzymic reactions involved in the cleavage of bonds 189-190 and 192-193 were first-order reactions. The four corresponding apparent rate constants were calculated by using a computer program. Excellent agreement was obtained between concentrations of four molecular species measured during the reaction period and those calculated by using the four apparent rate constants.     

Effect of Calcium Ions on Enteropeptidase Catalysis

The effects of calcium ions on hydrolysis of low molecular weight substrates catalyzed by different forms of enteropeptidase were studied. A method for determining activity of truncated enteropeptidase preparations lacking a secondary trypsinogen binding site and displaying low activity towards trypsinogen was developed using N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (Z-Lys-S-Bzl). The kinetic constants for hydrolysis of this substrate at pH 8.0 and 25 degrees C were determined for natural enteropeptidase (K(m) 59.6 microM, k(cat) 6660 min(-1), k(cat)/K(m) 111 microM(-1) x min(-1)), as well as for enteropeptidase preparation with deleted 118-783 fragment of the heavy chain (K(m) 176.9 microM, k(cat) 6694 min(-1), k(cat)/K(m) 37.84 microM(-1) x min(-1)) and trypsin (K(m) 56.0 microM, k(cat) 8280 min(-1), k(cat)/K(m) 147.86 microM(-1) x min(-1)). It was shown that the enzymes with trypsin-like primary active site display similar hydrolysis efficiency towards Z-Lys-S-Bzl. Calcium ions cause 3-fold activation of hydrolysis of the substrates of general type GD(4)K-X by the natural full-length enteropeptidase. In contrast, the hydrolysis of substrates with one or two Asp/Glu residues at P2-P3 positions is slightly inhibited by Ca2+. In the case of enteropeptidase light chain as well as the enzyme containing the truncated heavy chain (466-800 fragment), the activating effect of calcium ions was not detected for all the studied substrates. The results of hydrolysis experiments with synthetic enteropeptidase substrates GD(4)K-F(NO(2))G, G(5)DK-F(NO(2))G (where F(NO(2)) is p-nitrophenyl-L-phenylalanine residue), and GD(4)K-Nfa (where Nfa is beta-naphthylamide) demonstrate the possibility of regulation of undesired side hydrolysis using natural full-length enteropeptidase for processing chimeric proteins by means of calcium ions.     

Cobalt and the iron acquisition pathway: competition towards interaction with receptor 1

During iron acquisition by the cell, complete homodimeric transferrin receptor 1 in an unknown state (R1) binds iron-loaded human serum apotransferrin in an unknown state (T) and allows its internalization in the cytoplasm. T also forms complexes with metals other than iron. Are these metals incorporated by the iron acquisition pathway and how can other proteins interact with R1? We report here a four-step mechanism for cobalt(III) transfer from CoNtaCO(3)(2-) to T and analyze the interaction of cobalt-loaded transferrin with R1. The first step in cobalt uptake by T is a fast transfer of Co(3+) and CO(3)(2-) from CoNtaCO(3)(2-) to the metal-binding site in the C-lobe of T: direct rate constant, k(1)=(1.1+/-0.1) x 10(6) M(-1) s(-1); reverse rate constant, k(-1)=(1.9+/-0.6) x 10(6) M(-1) s(-1); and equilibrium constant, K=1.7+/-0.7. This step is followed by a proton-assisted conformational change of the C-lobe: direct rate constant, k(2)=(3+/-0.3) x 10(6) M(-1) s(-1); reverse rate constant, k(-2)=(1.6+/-0.3) x 10(-2) s(-1); and equilibrium constant, K(2a)=5.3+/-1.5 nM. The two final steps are slow changes in the conformation of the protein (0.5 h and 72 h), which allow it to achieve its final thermodynamic state and also to acquire second cobalt. The cobalt-saturated transferrin in an unknown state (TCo(2)) interacts with R1 in two different steps. The first is an ultra-fast interaction of the C-lobe of TCo(2) with the helical domain of R1: direct rate constant, k(3)=(4.4+/-0.6)x10(10) M(-1) s(-1); reverse rate constant, k(-3)=(3.6+/-0.6) x 10(4) s(-1); and dissociation constant, K(1d)=0.82+/-0.25 muM. The second is a very slow interaction of the N-lobe of TCo(2) with the protease-like domain of R1. This increases the stability of the protein-protein adduct by 30-fold with an average overall dissociation constant K(d)=25+/-10 nM. The main trigger in the R1-mediated iron acquisition is the ultra-fast interaction of the metal-loaded C-lobe of T with R1. This step is much faster than endocytosis, which in turn is much faster than the interaction of the N-lobe of T with the protease-like domain. This can explain why other metal-loaded transferrins or a protein such as HFE-with a lower affinity for R1 than iron-saturated transferrin but with, however, similar or higher affinities for the helical domain than the C-lobe-competes with iron-saturated transferrin in an unknown state towards interaction with R1.     

Multi-method global analysis of thermodynamics and kinetics in reconstitution of monellin

Accurate and precise determinations of thermodynamic parameters of binding are important steps toward understanding many biological mechanisms. Here, a multi-method approach to binding analysis is applied and a detailed error analysis is introduced. Using this approach, the binding thermodynamics and kinetics of the reconstitution of the protein monellin have been quantitatively determined in detail by simultaneous analysis of data collected with fluorescence spectroscopy, surface plasmon resonance and isothermal titration calorimetry at 25 degrees C, pH 7.0 and 150 mM NaCl. Monellin is an intensely sweet protein composed of two peptide chains that form a single globular domain. The kinetics of the reconstitution reaction are slow, with an association rate constant, k(on) of 8.8 x 10(3) M(-1) s(-1) and a dissociation rate constant, k(off) of 3.1 x 10(-4) s(-1). The equilibrium constant K(A) is 2.8 x 10(7) M(-1) corresponding to a standard free energy of association, DeltaG degrees , of -42.5 kJ/mol. The enthalpic component, DeltaH degrees , is -18.7 kJ/mol and the entropic contribution, DeltaS degrees , is 79.8 J mol(-1) K(-1) (-TDeltaS degrees = -23.8 kJ/mol). The association of monellin is therefore a bimolecular intra-protein association whose energetics are slightly dominated by entropic factors.     

Dynamics of interaction of vitamin C with some potent nitrovasodilators, S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and S-nitrosocaptopril (SNOCap), in aqueous solution

The reductive decomposition of both SNAP and SNOCap by ascorbate in aqueous solution (in the presence of EDTA) was thoroughly investigated. Nitric oxide (NO) release from the reaction occurs in an ascorbate concentration and pH dependent manner. Rates and hence NO release increased drastically with increasing pH, signifying that the most highly ionized form of ascorbate is the more reactive species. The experiments were monitored spectrophotometrically, and second-order rate constants calculated at 37 degrees C for the reduction of SNAP are k(b)=9.81+/-1.39 x 10(-3) M(-1) s(-1) and k(c)=662+/-38 M(-1) s(-1) and for SNOCap are k(b)=2.57+/-1.29 x 10(-2) M(-1) s(-1) and k(c)=49.7+/-1.3 M(-1) s(-1). k(b) and k(c) are the second-order rate constants via the ascorbate monoanion (HA-) and dianion (A2-) pathways, respectively. Activation parameters were also calculated and are DeltaHb++ =93+/-7 kJ mol(-1), DeltaSb++ =15+/-2 J K(-1) mol(-1) and DeltaHc++ =51+/-5 kJ mol(-1), DeltaSc++ =-28+/-3 J K(-1) mol(-1) with respect to the reactions involving SNAP. Those for the reaction between SNOCap and ascorbate were calculated to be DeltaHb++ =63+/-11 kJ mol(-1), DeltaSb++ =-71+/-20 J K(-1) mol(-1) and DeltaHc++ =103+/-7 kJ mol(-1), DeltaSc++ =118+/-8 J K(-1) mol(-1). The effect of Cu2+/Cu+ ions on the reductive decompositions of these S-nitrosothiols was also investigated in absence of EDTA. SNOCap exhibits relatively high stability at near physiological conditions (37 degrees C and pH 7.55) even in the presence of micromolar concentrations of Cu2+, with decomposition rate constant being 0.011 M(-1) s(-1) in comparison to SNAP which is known to be more susceptible to catalytic decomposition by Cu2+ (second-order rate constant of 20 M(-1) s(-1) at pH 7.4 and 25 degrees C). It was also observed that the reductive decomposition of SNAP is not catalyzed by alkali metal ions, however, there was an increase in rate as the ionic strength increases from 0.2 to 0.5 mol dm(-3) NaCl.     

A structure-based site-directed mutagenesis study on the neurolysin (EC 3.4.24.16) and thimet oligopeptidase (EC 3.4.24.15) catalysis

Neurolysin (EP24.16) and thimet oligopeptidase (EP24.15) are closely related metalloendopeptidases. Site-directed mutagenesis of Tyr(613) (EP24.16) or Tyr(612) (EP24.15) to either Phe or Ala promoted a strong reduction of k(cat)/K(M) for both enzymes. These data suggest the importance of both hydroxyl group and aromatic ring at this specific position during substrate hydrolysis by these peptidases. Furthermore, the EP24.15 A607G mutant showed a k(cat)/K(M) of 2x10(5) M(-1) s(-1) for the Abz-GFSIFRQ-EDDnp substrate, similar to that of EP24.16 (k(cat)/K(M)=3x10(5) M(-1) s(-1)) which contains Gly at the corresponding position; the wild type EP24.15 has a k(cat)/K(M) of 2.5x10(4) M(-1) s(-1) for this substrate.     

Modeling the haloperoxidases: reversible oxygen atom transfer between bromide ion and an oxo-Mn(V) porphyrin

The manganese meso-dimethylimidazolium porphyrin complex Mn(III)[TDMImP] reacted with HOBr/OBr(-) to generate the corresponding oxo-Mn(V)[TDMImP] species. The rate of this process accelerated with increasing pH. A forward rate constant, k(for), of 1.65x10(6)M(-1)s(-1) was determined at pH 8. Under these conditions, the oxo-Mn(V) species is short-lived and is transformed into the corresponding oxo-Mn(IV) complex. A first-order rate constant, k(obs), of 0.66 s(-1) was found for this reduction process at pH 8. The mechanism of this reduction process, which was dependent on bromide ion, appeared to proceed via an intermediate Mn(III)-O-Br complex. Thus, both a fast, reversible Mn(III)-O-Br bond heterolysis and a slower homolytic pathway occur in parallel in this system. The reverse oxidation reaction between oxo-Mn(V)[TDMImP] and bromide was investigated as a function of pH. The rate of this oxo-transfer reaction (k(rev)=1.4x10(3)M(-1)s(-1) at pH 8) markedly accelerated as the pH was lowered. The observed first-order dependence of the rate on [H(+)] indicates that the reactive species responsible for bromide oxidation is a protonated oxo-hydroxo complex and the stable species present in solution at high pH is dioxo-Mn(V)[TDMImP], [O=Mn(V)=O](-). The oxo-Mn(V) species retains nearly all of the oxidative driving force of the hypohalite. The equilibrium constant K(equi)=k(for)/k(rev) for the reversible process was determined at three different pH values (K(equi)=1.15x10(3) at pH 8) allowing the measurement of the redox potentials E of oxo-Mn(V)/Mn(III) (E=1.01 V at pH 8). The redox potential for this couple was extrapolated over the entire pH scale using the Nernst relationship and compared to those of the manganese 2- and 4-meso-N-methylpyridinium porphyrin couples oxo-Mn(V)[2-TMPyP]/Mn(III)[2-TMPyP], oxo-Mn(V)[4-TMPyP]/Mn(III)[4-TMPyP], OBr(-)/Br(-) and H(2)O(2)/H(2)O. Notably, the redox potential of oxo-Mn(V)/Mn(III) for the imidazolium porphyrin approaches that of H(2)O(2)/H(2)O at low pH.     

Mechanistic origins of the substrate selectivity of serine proteases

Serine proteases catalyze the hydrolysis of amide bonds of their protein and peptide substrates through a mechanism involving the intermediacy of an acyl-enzyme. While the rate constant for formation of this intermediate, k(2), shows a dramatic dependence on peptide chain length, the rate constant for the intermediate's hydrolysis is relatively insensitive to chain length. To probe the mechanistic origins of this phenomenon, we determined temperature dependencies and solvent isotope effects for the alpha-chymotrypsin-catalyzed hydrolysis of Suc-Phe-pNA (K(s) = 1 mM, k(2) = 0.04 s(-)(1), and k(3) = 11 s(-)(1)), Suc-Ala-Phe-pNA (K(s) = 4 mM, k(2) = 0.9 s(-)(1), and k(3) = 42 s(-)(1)), and Suc-Ala-Ala-Pro-Phe-pNA (K(s) = 0.1 mM, k(2) = 98 s(-)(1), and k(3) = 71 s(-)(1)). We found that while the van't Hoff plots for K(s) and the Eyring plots for k(3) are linear for all three reactions, the Eyring plots for k(2) are convex, indicating that the process governed by k(2) is complex, possibly involving a coupling between active site chemistry and protein conformational isomerization. This interpretation is strengthened by solvent isotope effects on k(2) that are largely temperature-independent. Furthermore, the dependence of k(2) on peptide length is manifested entirely in the enthalpy of activation, suggesting a mechanism of catalysis by distortion. Taken together, this analysis of acylation suggests that extended substrates which can engage in subsite interactions are able to efficiently trigger the coupling mechanism between chemistry and a conformational isomerization that distorts the substrate and thereby promotes nucleophilic attack.     

Cloning and functional expression of a rodent brain cDNA encoding a novel protein with agmatinase activity, but not belonging to the arginase family

A rat brain cDNA encoding for a novel protein with agmatinase activity was cloned and functionally expressed. The protein was expressed as a histidine-tagged fusion product with a molecular weight of about 63 kDa. Agmatine hydrolysis was strictly dependent on Mn(2+); K(m) and k(cat) values were 2.5+/-0.2 mM and 0.8+/-0.2 s(-1), respectively. The product putrescine was a linear competitive inhibitor (K(i)=5+/-0.5 mM). The substrate specificity, metal ion requirement and pH optimum (9.5) coincide with those reported for Escherichia coli agmatinase, the best characterized of the agmatinases. However, as indicated by the k(cat)/K(m) (320 M(-1)s(-1)), the recombinant protein was about 290-fold less efficient than the bacterial enzyme. The deduced amino sequence revealed great differences with all known agmatinases, thus excluding the protein from the arginase family. It was, however, highly identical (>85%) to the predicted sequences for fragments of hypothetical or unnamed LIM domain-containing proteins. As a suggestion, the agmatinase activity is adscribed to a protein with an active site that promiscuously catalyze a reaction other than the one it evolved to catalyze.     

Importance of product inhibition in the kinetics of the acylase hydrolysis reaction by differential stopped flow microcalorimetry

The hydrolysis of N-acetyl-L-methionine, N-acetylglycine, N-acetyl-L-phenylalanine, and N-acetyl-L-alanine at 298.35K by porcine kidney acylase I (EC 3.5.1.14) was monitored by the heat released upon mixing of the substrate and enzyme in a differential stopped flow microcalorimeter. Values for the Michaelis constant (K(m)) and the catalytic constant (k(cat)) were determined from the progress of the reaction curve employing the integrated form of the Michaelis-Menten equation for each reaction mixture. When neglecting acetate product inhibition of the acylase, values for k(cat) were up to a factor of 2.3 larger than those values determined from reciprocal initial velocity-initial substrate concentration plots for at least four different reaction mixtures. In addition, values for K(m) were observed to increase linearly with an increase in the initial substrate concentration. When an acetate product inhibition constant of 600+/-31M(-1), determined by isothermal titration calorimetry, was used in the progress curve analysis, values for K(m) and k(cat) were in closer agreement with their values determined from the reciprocal initial velocity versus initial substrate concentration plots. The reaction enthalpies, Delta(r)H(cal), which were determined from the integrated heat pulse per amount of substrate in the reaction mixture, ranged from -4.69+/-0.09kJmol(-1) for N-acetyl-L-phenylalanine to -1.87+/-0.23kJmol(-1) for N-acetyl-L-methionine.     

Properties of urease immobilized on the functional organic silica surface

The paper deals with kinetics of the urea hydrolysis by microbial-origin urease dissolved and immobilized on the organic silica surface. It is shown that hydrolysis kinetics for soluble urease is described by the Michaelis-Menten equation until the concentration of urea reaches 1 M. Two fractions differing in the Michaelis constant are revealed for silochrome immobilized urease. The rate of urea hydrolysis by native and immobilized urease was studied depending on the pH value in presence of the substrate in the 1 M and 5 mM concentration. The hydrolysis rate of 1 M urea in the buffer-free solution by silochrome-immobilized urease is practically independent of pH within 4.5-6.5. Application of a 2.5 mM phosphate-citrate buffer as a solvent causes an increase in the hydrolysis rate within this pH range. For a soluble urease the 1 M urea hydrolysis rate dependence on pH is ordinary at pH 5.8-6.0. If the substrate concentration is 5 mM, the pH-dependences for the rate of the urea hydrolysis by silochrome- and aerosil-immobilized urease are close and at pH above 6.0 coincide with those for a soluble enzyme. The found differences in the properties of soluble and immobilized ureases are explained by the substrate and reaction products diffusion.