Two-photon excitation and photolysis by pulsed laser illumination modelled by spatially non-uniform reactions with simultaneous diffusion

Two-photon absorption in the focus of a pulsed laser has the potential for localized photolysis of caged compounds, generating high concentrations of neurotransmitters, hormones and messengers. The concentrations of cage, intermediates and products in the femtolitre focal volume depend on reaction rates and diffusional exchange with the external volume. This problem of reaction with diffusion was analysed with analytical and numerical methods to determine simple relations between parameters useful in the design and interpretation of experiments. The diffraction-limited laser spot is approximated well by a sphere, radius A, in diffusional exchange with either an infinite uniform medium, representing extracellular photolysis, or within a non-permeable sphere, a "cell" of radius B, representing intracellular photolysis. Photolysis is modelled as sequential irreversible reactions, with either the excitation step alone, rate constant k(e), or with a subsequent "dark" reaction, rate constant k(p). For extracellular photolysis, steady-state depletion of a cage averaged in a spherical spot increases hyperbolically with k(e) with half-maximum depletion at k(e) = K0.5 = 2.5 D/A2, where D is the diffusion coefficient. With measured parameters for spot size A = 0.3 microm and diffusion D = 800 microm2/s, K0.5 = 22,200 s(-1). The optimal exposure for localized photolysis is the characteristic diffusion time tau = A2/D, 113 micros in this example, and is the time taken to reach 57% of steady state in the diffusion-limited case. In the two-step model, with excitation and "dark" reaction steps, rate constants both exceeding K0.5 are necessary to generate 50% of maximal product concentration in the illuminated volume. High concentrations of photolysis products depend particularly on a high excitation rate constant (k(e) > K0.5), and localization of the products requires fast dark reactions (k(p) > K0.5). If products diffuse faster than the cage, their steady-state concentrations are decreased, and concentration transients may occur. For localized intracellular photolysis, the duration of exposure that generates product concentration at the cell boundary, B, less than 10% of the spot concentration should be shorter than 0.043(B/A)3tau, and is determined by diffusion.     

Kinetics of substrate transglycosylation by glycoside hydrolase family 3 glucan (1-->3)-beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium

To elucidate the interaction between substrate inhibition and substrate transglycosylation of retaining glycoside hydrolases (GHs), a steady-state kinetic study was performed for the GH family 3 glucan (1-->3)-beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium, using laminarioligosaccharides as substrates. When laminaribiose was incubated with the enzyme, a transglycosylation product was detected by thin-layer chromatography. The product was purified by size-exclusion chromatography, and was identified as a 6-O-glucosyl-laminaribiose (beta-D-Glcp-(1-->6)-beta-D-Glcp-(1-->3)-D-Glc) by 1H NMR spectroscopy and electrospray ionization mass spectrometry analysis. In steady-state kinetic studies, an apparent decrease of laminaribiose hydrolysis was observed at high concentrations of the substrate, and the plots of glucose production versus substrate concentration were thus fitted to a modified Michaelis-Menten equation including hydrolytic and transglycosylation parameters (K(m), K(m2), k(cat), k(cat2)). The rate of 6-O-glucosyl-laminaribiose production estimated by high-performance anion-exchange chromatography coincided with the theoretical rate calculated using these parameters, clearly indicating that substrate inhibition of this enzyme is fully explained by substrate transglycosylation. Moreover, when K(m), k(cat), and affinity for glucosyl-enzyme intermediates (K(m2)) were estimated for laminarioligosaccharides (DP=3-5), the K(m) value of laminaribiose was approximately 5-9 times higher than those of the other oligosaccharides (DP=3-5), whereas the K(m2) values were independent of the DP of the substrates. The kinetics of transglycosylation by the enzyme could be well interpreted in terms of the subsite affinities estimated from the hydrolytic parameters (K(m) and k(cat)), and a possible mechanism of transglycosylation is proposed.     

Catalytic properties of dihydroorotate dehydrogenase from Saccharomyces cerevisiae: studies on pH, alternate substrates, and inhibitors

Yeast dihydroorotate dehydrogenase (DHOD) was purified 2800-fold to homogeneity from its natural source. Its sequence is 70% identical to that of the Lactococcus lactis DHOD (family IA) and the two active sites are nearly the same. Incubations of the yeast DHOD with dideuterodihydroorotate (deuterated in the positions eliminated in the dehydrogenation) as the donor and [14C]orotate as the acceptor revealed that the C5 deuteron exchanged with H2O solvent at a rate equal to the 14C exchange rate, whereas the C6 deuteron was infrequently exchanged with H2O solvent, thus indicating that the C6 deuteron of the dihydroorotate is sticky on the flavin cofactor. The pH dependencies of the steady-state parameters (k(cat) and k(cat)/Km) are similar, indicating that k(cat)/Km reports the productive binding of substrate, and the parameters are dependent on the donor-acceptor pair. The lower pKa values for k(cat) and k(cat)/Km observed for substrate dihydroorotate (around 6) in comparison to the values determined for dihydrooxonate (around 8) suggest that the C5 pro S hydrogen atom of dihydroorotate (but not the analogous hydrogen of dihydrooxonate), which is removed in the dehydrogenation, assists in lowering the pKa of the active site base (Cys133). The pH dependencies of the kinetic isotope effects on steady-state parameters observed for the dideuterated dihydroorotate are consistent with the dehydrogenation of substrate being rate limiting at low pH values, with a pKa value approximating that assigned to Cys133. Electron acceptors with dihydroorotate as donor were preferred in the following order: ferricyanide (1), DCPIP (0.54), Qo (0.28), fumarate (0.15), and O2 (0.035). Orotate inhibition profiles versus varied concentrations of dihydroorotate with ferricyanide or O2 as acceptors suggest that both orotate and dihydroorotate have significant affinities for the reduced and oxidized forms of the enzyme.     

The effect of methanol and dioxan on the rates of the β-galactosidase-catalysed hydrolyses of some β-d-galactopyranosides: rate-limiting degalactosylation. The pH-dependence of galactosylation and degalactosylation

1. The effect of methanol on the beta-galactosidase-catalysed hydrolysis of some nitrophenyl beta-d-galactopyranosides has been studied under steady-state conditions. 2. The initial fractional rate of increase of k(cat.) as a function of methanol concentration with 2,4- and 3,5-dinitrophenyl beta-d-galactopyranosides, but not with the other substrates studied, indicated that degalactosylation of the enzyme was rate-limiting. 3. The decrease in k(cat.) at high methanol concentrations for these substrates is considered to arise from causes other than galactosylation becoming rate-limiting. 4. Both galactosylation and degalactosylation of the enzyme require protonation of a group of pK(a) approx. 9.     

Studies on rat liver plasma membrane. Altered protein and phospholipid metabolism after injection of D-galactosamine.

The displacement of NADH from cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by using NAD+, 1,10-phenanthroline, ADP-ribose, deamino-NAD+ and pyridine-3-aldehyde-adenine dinucleotide as displacing agents, by following the decrease in fluorescence as a function of time. The data obtained could be fitted by assuming two first-order processes were occurring, a faster process with an apparent rate constant of 0.85 +/- 0.20 s-1 and a relative amplitude of 60 +/- 10% and a slower process with an apparent rate constant of 0.20 +/- 0.05 s-1 and a relative amplitude of 40 +/- 10% (except for pyridine-3-aldehyde-adenine dinucleotide, where the apparent rate constant for the slow process was 0.05 s-1). The displacement rates did not change significantly when the pH was varied from 6.0 to 9.0. Kinetic data are also reported for the dependence of the rate of binding of NADH to the enzyme on the total concentration of NADH. Detailed arguments are presented based on the isolation and purification procedures, the equilibrium coenzyme-binding studies and the kinetic data, which lead to the following model for the release of NADH from the enzyme: (formula: see article). The parameters that best fit the data are: k + 1 = 0.2 s-1; k - 1 = 0.05 s-1; k + 2 = 0.8 s-1 and k - 2 = 5 X 10(5)litre-mol-1-s-1. The slow phase of the NADH release is similar to the steady-state turnover number for substrates such as acetaldehyde and propionaldehyde and appears to contribute significantly to the limitation of the steady-state rate.     

Parameterization of individual-based models: comparisons with deterministic mean-field models

The relating of deterministic, mean-field models into network models, where epidemic spread occurs between interconnected susceptible and infectious individuals or populations, requires careful consideration. Here, we discuss models that consider differently the manner in which contact rate and infectiousness change over time, with different algorithms suitable for different underlying processes. Though these models give coincidental results to the mean-field in the case of large, highly connected networks, the results when sparsely connected networks are considered may differ. Different subsets of the parameters from the mean-field epidemic (R(0), generation time, infectiousness, etc.) are preserved in each case. Despite these differences, simulated epidemics generated under some model architectures are insensitive to the average degree of contact amongst nodes, k. Model-based estimates of k may be model dependent, and must therefore be viewed with caution.     

Catalytic properties of porcine pancreatic elastase: a steady-state and pre-steady-state study

Pre-steady-state and steady-state kinetics for the p.p. elastase-catalysed hydrolysis of ZAlaONp, one of the most favourable substrates for this serine protease, have been studied between pH 4.0 and 8.0. The results are consistent with the minimum three-step mechanism: (formula; see text) Under pre-steady-state conditions, where [E0] much greater than [S0], the values of the dissociation constant of the E X S complex (Ks = k-1/k+1) and of the individual rate constants for the catalytic steps (k+2 and k+3) have been determined over the whole pH range explored. Under steady-state conditions, where [S0] much greater than [E0], the values of kcat and Km have been obtained over the same pH range. The pH profiles of k+2, k+3, k+2/Ks, kcat, kcat/Km reflect the ionization of a group, probably His57, with a pKa value of 6.85 +/- 0.10. The values of Ks and Km are pH independent. The steady-state parameters for the p.p. elastase-catalysed hydrolysis of a number of p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids have been also determined between pH 4.0 and 8.0 and compared with those of b.beta-trypsin and b.alpha-chymotrypsin. For all the substrates examined the acylation step (k+2) is rate limiting in the p.p. elastase catalysis, between pH 4.0 and 8.0. The different catalytic behaviours of p.p. elastase, b.beta-trypsin and b.alpha-chymotrypsin are consistent with the known three-dimensional structures of these serine proteases.     

Product release is rate-limiting in the activation of the prodrug 5-fluorocytosine by yeast cytosine deaminase

Yeast cytosine deaminase (yCD), a zinc metalloenzyme, catalyzes the hydrolytic deamination of cytosine to uracil. The enzyme is of great biomedical interest because it also catalyzes the deamination of the prodrug 5-fluorocytosine (5FC) to form the anticancer drug 5-fluorouracil (5FU). yCD/5FC is one of the most widely used enzyme/prodrug combinations for gene-directed enzyme prodrug therapy for the treatment of cancers. A pH indicator assay has been developed for the measurement of the steady-state kinetic parameters for the deamination reaction. Transient kinetic studies have shown that the product release is a rate-limiting step in the activation of the prodrug 5FC by yCD. The rate constant of the chemical step for the forward reaction (250 s(-)(1)) is approximately 8 times that of the product release (31 s(-)(1)) and approximately 15 times k(cat) (17 s(-)(1)). The transient kinetic results are consistent with those of the steady-state kinetic analysis in the sense that the k(cat) and K(m) values calculated from the rate constants determined by the transient kinetic analysis are in close agreement with those measured by the steady-state kinetic analysis. NMR experiments have demonstrated that free 5FU is in slow exchange with its complex with yCD but has a low affinity for yCD. The transient kinetic and NMR results together suggest that the release of 5FU is rate-limiting in the activation of the prodrug 5FC by yCD and may involve multiple steps.     

Reaction of vascular adhesion protein-1 (VAP-1) with primary amines: mechanistic insights from isotope effects and quantitative structure-activity relationships

Human vascular adhesion protein-1 (VAP-1) is an endothelial copper-dependent amine oxidase involved in the recruitment and extravasation of leukocytes at sites of inflammation. VAP-1 is an important therapeutic target for several pathological conditions. We expressed soluble VAP-1 in HEK293 EBNA1 cells at levels suitable for detailed mechanistic studies with model substrates. Using the model substrate benzylamine, we analyzed the steady-state kinetic parameters of VAP-1 as a function of solution pH. We found two macroscopic pK(a) values that defined a bell-shaped plot of turnover number k(cat,app) as a function of pH, representing ionizable groups in the enzyme-substrate complex. The dependence of (k(cat)/K(m))(app) on pH revealed a single pK(a) value (～9) that we assigned to ionization of the amine group in free benzylamine substrate. A kinetic isotope effect (KIE) of 6 to 7.6 on (k(cat)/K(m))(app) over the pH range of 6 to 10 was observed with d(2)-benzylamine. Over the same pH range, the KIE on k(cat) was found to be close to unity. The unusual KIE values on (k(cat)/K(m))(app) were rationalized using a mechanistic scheme that includes the possibility of multiple isotopically sensitive steps. We also report the analysis of quantitative structure-activity relationships (QSAR) using para-substituted protiated and deuterated phenylethylamines. With phenylethylamines we observed a large KIE on k(cat,app) (8.01 ± 0.28 with phenylethylamine), indicating that C-H bond breakage is limiting for 2,4,5-trihydroxyphenylalanine quinone reduction. Poor correlations were observed between steady-state rate constants and QSAR parameters. We show the importance of combining KIE, QSAR, and structural studies to gain insight into the complexity of the VAP-1 steady-state mechanism.     

<Emphasis Type="Italic">k</Emphasis><Subscript>L</Subscript><Emphasis Type="Italic">a</Emphasis> determination: comparative study for a gas mass balance method

The determination of k(L) a by a gas balance method coupled with sulphite oxidation is compared for three kinds of processes (stirred tank, bubble column and fixed-bed column reactors) with a gassing-in and with a classical chemical sulphite oxidation method. The mathematical relations required for the determination of the k(L) a value are detailed. In coalescing gas-liquid conditions, the values calculated by the three methods are shown to be comparable. The gas balance method is more rapid than either the steady-state gassing-in or the chemical sulphite reaction rate measurement methods. It is also well adapted for three-phase systems (gas-liquid-solid) in which the non-coalescing effects of sulphite solution are reduced by solid interferences.     

Photophysics of the fluorescent Ca2+ indicator Fura-2.

The photophysics of the complex forming reaction of Ca2+ and Fura-2 are investigated using steady-state and time-resolved fluorescence measurements. The fluorescence decay traces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution with BAPTA as Ca2+ buffer: k01 = 1.2 x 10(9)s-1, k21 = 1.0 x 10(11) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 2.2 x 10(7) s-1, and with EGTA as Ca2+ buffer: k01 = 1.4 x 10(9) s-1, k21 = 5.0 x 10(10) M-1 s-1, k02 = 5.5 x 10(8) s-1, k12 = 3.2 x 10(7) s-1. k01 and k02 denote the respective deactivation rate constants of the Ca2+ free and bound forms of Fura-2 in the excited state. k21 represents the second-order rate constant of binding of Ca2+ and Fura-2 in the excited state, whereas k12 is the first-order rate constant of dissociation of the excited Ca2+:Fura-2 complex. The ionic strength of the solution was shown not to influence the recovered values of the rate constants. From the estimated values of k12 and k21, the dissociation constant K*d in the excited state was calculated. It was found that in EGTA Ca2+ buffer pK*d (3.2) is smaller than pKd (6.9) and that there is negligible interference of the excited-state reaction with the determination of Kd and [Ca2+] from fluorimetric titration curves. Hence, Fura-2 can be safely used as an Ca2+ indicator. From the obtained fluorescence decay parameters and the steady-state excitation spectra, the species-associated excitation spectra of the Ca2+ free and bound forms of Fura-2 were calculated at intermediate Ca2+ concentrations.     

Modification of A-stat for the characterization of microorganisms

Two novel modifications of continuous culture with gradual change of dilution rate (A-stat): D-stat and auxo-accelerostat were evaluated in the studies of the effect of changing individual environmental parameters (T, pH, pO(2), substrate concentration, etc.) on growth characteristics of different microorganisms. Common for those cultivation methods is that one environmental parameter is programmed to change with constant change rate (change-stat) while the others are kept constant or in the range not affecting the growth characteristics. The environment response growth curves were obtained starting with chemostat (in A-stat and D-stat) or auxostat (in auxo-accelerostat) steady-state cultures followed by change of set-point value of the desired cultivation parameter. Physiological studies of Saccharomyces sp. and Lactococcus lactis were combined with validation of the different modifications of the A-stat method based on well-known cultivation techniques: chemostat, pH-auxostat, pO(2)-auxostat CO(2)-auxostat and fed-batch. The auxo-accelerostat was shown to be very efficient for cell characterization and dynamic studies in growth environments with excess of essential substrates. Choosing the rate of change of environmental parameters was shown to be critical in comparative physiological studies of microorganisms.     

Isotopic probes yield microscopic constants: separation of binding energy from catalytic efficiency in the bovine plasma amine oxidase reaction

Bovine plasma amine oxidase catalyzes the oxidative deamination of primary amines. The reaction can be viewed as two half-reactions: enzyme reduction by substrate followed by enzyme reoxidation by dioxygen. Anaerobic stopped-flow kinetic measurements of the first half-reaction indicate very large deuterium isotope effects for benzylamine, m-tyramine, and dopamine, Dk = 13.5 +/- 1.3, which are ascribed to an intrinsic isotope effect. From the insensitivity of these isotope effects to amine concentration, stopped-flow data provide substrate dissociation constants, K1, and rate constants for the C-H bond cleavage step, k3, directly. Steady-state isotope effects have also been measured for benzylamine and six ring-substituted phenethylamines. Whereas a small range of values for kcat, 0.38-1.2 s-1, and Dkcat, 5.4-8.8, is observed, kcat/Km = 1.3 X 10(2) to 3.8 X 10(4) M-1 S-1 and D(kcat/Km) = 5.6-16.1 indicate a marked effect of ring substituent. As described earlier [Miller, S., & Klinman, J.P. (1982) Methods Enzymol. 87, 711], the availability of an intrinsic isotope effect for an enzymatic reaction permits calculation of microscopic constants from steady-state data. By employment of a minimal mechanism for bovine plasma amine oxidase involving a single precatalytic and multiple postcatalytic enzyme-substrate complexes, equations have been derived that allow calculation of k3 and K1 when DKeq congruent to 1 less than Dk. Unexpectedly, in the case of K1, we have shown that this parameter can be calculated from steady-state parameters without the requirement for an intrinsic isotope effect. This result should have general application to both ping-pong and sequential ternary-complex enzyme mechanisms. Of significance for future applications of steady-state isotope effects to the calculation of microscopic constants, values for K1 and k3 derived from steady-state parameters and single turnover measurements indicate excellent agreement. Compilation of parameters among six ring-substituted phenethylamines reveals alteration in delta G for enzyme-substrate complex formation by 2.8 kcal/mol, together with an essentially invariant rate constant for C-H bond activation. A detailed discussion of the relevance of these findings to the interrelationship of binding energy and catalytic efficiency in enzyme reactions is presented.     

Rapid kinetic and isotopic studies on dialkylglycine decarboxylase

The two half-reactions of the pyridoxal 5'-phosphate (PLP)-dependent enzyme dialkylglycine decarboxylase (DGD) were studied individually by multiwavelength stopped-flow spectroscopy. Biphasic behavior was found for the reactions of DGD-PLP, consistent with two coexisting conformations observed in steady-state kinetics [Zhou, X., and Toney, M. D. (1998) Biochemistry 37, 5761--5769]. The half-reaction kinetic parameters depend on alkali metal ion size in a manner similar to that observed for steady-state kinetic parameters. The fast phase maximal rate constant for the 2-aminoisobutyrate (AIB) decarboxylation half-reaction with the potassium form of DGD-PLP is 25 s(-1), while that for the transamination half-reaction between DGD-PMP and pyruvate is 75 s(-1). The maximal rate constant for the transamination half-reaction of the potassium form of DGD-PLP with L-alanine is 24 s(-1). The spectral data indicate that external aldimine formation with either AIB or L-alanine and DGD-PLP is a rapid equilibrium process, as is ketimine formation from DGD-PMP and pyruvate. Absorption ascribable to the quinonoid intermediate is not observed in the AIB decarboxylation half-reaction, but is observed in the dead-time of the stopped-flow in the L-alanine transamination half-reaction. The [1-(13)C]AIB kinetic isotope effect (KIE) on k(cat) for the steady-state reaction is 1.043 +/- 0.003, while a value of 1.042 +/- 0.009 was measured for the AIB half-reaction. The secondary KIE measured for the AIB decarboxylation half-reaction with [C4'-(2)H]PLP is 0.92 +/- 0.02. The primary [2-(2)H]-L-alanine KIE on the transamination half-reaction is unity. Small but significant solvent KIEs are observed on k(cat) and k(cat)/K(M) for both substrates, and the proton inventories are linear in each case. NMR measurements of C2--H washout vs product formation give ratios of 105 and 14 with L-alanine and isopropylamine as substrates, respectively. These results support a rate-limiting, concerted C alpha-decarboxylation/C4'-protonation mechanism for the AIB decarboxylation reaction, and rapid equilibrium quinonoid formation followed by rate-limiting protonation to the ketimine intermediate for the L-alanine transamination half-reaction. Energy profiles for the two half-reactions are constructed.     

Trypsin-like serine proteinase action: determination of the catalytic parameters KS, k+2 and k/3 under conditions where the substrate exceeds the enzyme concentration

A method for the determination of the catalytic parameters Ks, k+2 and k+3 describing trypsin-like serine proteinase action has been developed from the quantitative analysis of the kinetics of hydrolysis of two specific chromogenic substrates, N-alpha-carbobenzoxy-L-arginine p-nitrophenyl ester and N-alpha-carbobenzoxy-L-lysine p-nitrophenyl ester, catalyzed by porcine pancreatic betta-kallikrein B, bovine betta-trypsin and human urokinase (Mr 54,000 species), under conditions where the concentration of the substrate exceeds that of the enzyme. Value of Ks, k+2 and k+3 have been estimated from the effect of substrate concentration on the apparent first-order rate constant of the time-course of the burst phase of p-nitrophenol release preceding the steady-state reaction.     

Cross-bridge versus thin filament contributions to the level and rate of force development in cardiac muscle

In striated muscle thin filament activation is initiated by Ca(2+) binding to troponin C and augmented by strong myosin binding to actin (cross-bridge formation). Several lines of evidence have led us to hypothesize that thin filament properties may limit the level and rate of force development in cardiac muscle at all levels of Ca(2+) activation. As a test of this hypothesis we varied the cross-bridge contribution to thin filament activation by substituting 2 deoxy-ATP (dATP; a strong cross-bridge augmenter) for ATP as the contractile substrate and compared steady-state force and stiffness, and the rate of force redevelopment (k(tr)) in demembranated rat cardiac trabeculae as [Ca(2+)] was varied. We also tested whether thin filament dynamics limits force development kinetics during maximal Ca(2+) activation by comparing the rate of force development (k(Ca)) after a step increase in [Ca(2+)] with photorelease of Ca(2+) from NP-EGTA to maximal k(tr), where Ca(2+) binding to thin filaments should be in (near) equilibrium during force redevelopment. dATP enhanced steady-state force and stiffness at all levels of Ca(2+) activation. At similar submaximal levels of steady-state force there was no increase in k(tr) with dATP, but k(tr) was enhanced at higher Ca(2+) concentrations, resulting in an extension (not elevation) of the k(tr)-force relationship. Interestingly, we found that maximal k(tr) was faster than k(Ca), and that dATP increased both by a similar amount. Our data suggest the dynamics of Ca(2+)-mediated thin filament activation limits the rate that force develops in rat cardiac muscle, even at saturating levels of Ca(2+).     

Steady-state and pre-steady-state kinetic analysis of halopropane conversion by a rhodococcus haloalkane dehalogenase

Haloalkane dehalogenase from Rhodococcus rhodochrous NCIMB 13064 (DhaA) catalyzes the hydrolysis of carbon-halogen bonds in a wide range of haloalkanes. We examined the steady-state and pre-steady-state kinetics of halopropane conversion by DhaA to illuminate mechanistic details of the dehalogenation pathway. Steady-state kinetic analysis of DhaA with a range of halopropanes showed that bromopropanes had higher k(cat) and lower K(M) values than the chlorinated analogues. The kinetic mechanism of dehalogenation was further studied using rapid-quench-flow analysis of 1,3-dibromopropane conversion. This provided a direct measurement of the chemical steps in the reaction mechanism, i.e., cleavage of the carbon-halogen bond and hydrolysis of the covalent alkyl-enzyme intermediate. The results lead to a minimal mechanism consisting of four main steps. The occurrence of a pre-steady-state burst, both for bromide and 3-bromo-1-propanol, suggests that product release is rate-limiting under steady-state conditions. Combining pre-steady-state burst and single-turnover experiments indicated that the rate of carbon-bromine bond cleavage was indeed more than 100-fold higher than the steady-state k(cat). Product release occurred with a rate constant of 3.9 s(-1), a value close to the experimental k(cat) of 2.7 s(-1). Comparing the kinetic mechanism of DhaA with that of the corresponding enzyme from Xanthobacter autotrophicus GJ10 (DhlA) shows that the overall mechanisms are similar. However, whereas in DhlA the rate of halide release represents the slowest step in the catalytic cycle, our results suggest that in DhaA the release of 3-bromo-1-propanol is the slowest step during 1,3-dibromopropane conversion.     

Reaction of ascorbic acid with cytochrome b561. Concerted electron and proton transfer.

Rate constants for reduction of cytochrome b561 by internal ascorbate (k0A) and oxidation by external ferricyanide (k1F) were determined as a function of pH from rates of steady-state electron transfer across chromaffin-vesicle membranes. The pH dependence of electron transfer from cytochrome b561 to ferricyanide (k1F) may be attributed to the pH dependence of the membrane surface potential. The rate constant for reduction by internal ascorbate (k0A), like the previously measured rate constant for reduction by external ascorbate (k-1A), is not very pH-dependent and is not consistent with reduction of cytochrome b561 by the ascorbate dianion. The rate at which ascorbate reduces cytochrome b561 is orders of magnitude faster than the rate at which it reduces cytochrome c, despite the fact that midpoint reduction potentials favor reduction of cytochrome c. Moreover, the rate constant for oxidation of cytochrome b561 by ferricyanide (k1F) is smaller than the previously measured rate constant for oxidation by semidehydroascorbate, despite the fact that ferricyanide has a higher midpoint reduction potential. These results may be reconciled by a mechanism in which electron transfer between cytochrome b561 and ascorbate/semidehydroascorbate is accelerated by concerted transfer of a proton. This may be a general property of biologically significant electron transfer reactions of ascorbic acid.     

Molecular functions of conserved aspects of the GHMP kinase family

The sequences and three-dimensional structures of the galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase (GHMP) family were compared to identify highly conserved surface residues. The functions of these solvent-accessible residues were assessed by determining the effects of their substitution, via mutagenesis, on the initial-rate parameters of a representative member of the GHMP kinase family, phosphomevalonate kinase from Streptococcus pneumoniae. What emerges from this study is a profile of the conserved surface-linked functions of the family. Certain substitutions produce highly selective effects on the steady-state affinity of a particular substrate, while one residue, Asp150, appears to be a pure k(cat) effector. Substitutions elsewhere affect multiple initial-rate parameters with varying, and sometimes compensatory, patterns. An alpha-helix that repositions during catalysis was substituted along its length to assess how its different segments contribute to catalysis-the substrate-proximal edge of the helix affects ATP recognition and k(cat), while the distal edge affects recognition of both substrates without affecting turnover. GHMP kinase mutations at the conserved surface residues corresponding to Ser291 and Ala293 in phosphomevalonate kinase are linked to mevalonic acid deficiency, which can lead to early fatality, and galactokinase deficiency, which causes cataracts. Our results suggest that the molecular basis for this particular galactokinase deficiency is an increase in the K(m) for galactose.     

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.