Triosephosphate isomerase catalysis is diffusion controlled. Appendix: Analysis of triose phosphate equilibria in aqueous solution by 31P NMR

The rates of the forward and reverse reactions of triosephosphate isomerase catalyzed by the wild-type and by a sluggish mutant enzyme have been studied in the absence and the presence of several viscosogenic agents. For the mutant enzyme, the kcat for which is some 10(3) times less than that for the wild-type enzyme, the value of kcat/Km with glyceraldehyde phosphate as substrate is almost unaffected by the presence of sucrose or glycerol, even though the concentration of the aldehyde form of the substrate is smaller because of hemiacetal formation. [The nature and relative amounts of the various forms of triose phosphate present in solution (free carbonyl forms, hydrates, dimers, hemiacetal adducts) have been evaluated by 31P NMR and are presented in the Appendix.] The viscosogenic agents cause the substrate to bind more tightly to the enzyme, roughly compensating for the lower substrate concentration. With dihydroxyacetone phosphate as substrate, the values of kcat/Km for the mutant enzyme increase with the addition of viscosogenic agent, consistent with the tighter binding of substrate without (in this case) any concomitant loss due to hemiketal formation. These results for the mutant enzyme (known to be limited in rate by an enolization step in the catalytic mechanism) can be used to interpret the behavior of the wild-type enzyme. Plots of the relative values of kcat/Km for catalysis by the wild-type enzyme (normalized with the corresponding data for the mutant enzyme) against the relative viscosity have slopes close to unity, as predicted by the Stokes-Einstein equation for a cleanly diffusive process. In the presence of polymeric viscosogenic additives such as poly(ethylene glycol), polyacrylamide, or ficoll, no effect on kcat/Km is seen for the wild-type enzyme, consistent with the expectation that molecular diffusion rates are unaffected by the macroviscosity and are only slowed by the presence of smaller agents that raise the microviscosity. These results show that the reaction catalyzed by the wild-type triosephosphate isomerase is limited by the rate at which glyceraldehyde phosphate encounters, or departs from, the active site.     

Investigation of the functional role of tryptophan-22 in Escherichia coli dihydrofolate reductase by site-directed mutagenesis

We have applied site-directed mutagenesis methods to change the conserved tryptophan-22 in the substrate binding site of Escherichia coli dihydrofolate reductase to phenylalanine (W22F) and histidine (W22H). The crystal structure of the W22F mutant in a binary complex with the inhibitor methotrexate has been refined at 1.9-A resolution. The W22F difference Fourier map and least-squares refinement show that structural effects of the mutation are confined to the immediate vicinity of position 22 and include an unanticipated 0.4-A movement of the methionine-20 side chain. A conserved bound water-403, suspected to play a role in the protonation of substrate DHF, has not been displaced by the mutation despite the loss of a hydrogen bond with tryptophan-22. Steady-state kinetics, stopped-flow kinetics, and primary isotope effects indicate that both mutations increase the rate of product tetrahydrofolate release, the rate-limiting step in the case of the wild-type enzyme, while slowing the rate of hydride transfer to the point where it now becomes at least partially rate determining. Steady-state kinetics show that below pH 6.8, kcat is elevated by up to 5-fold in the W22F mutant as compared with the wild-type enzyme, although kcat/Km(dihydrofolate) is lower throughout the observed pH range. For the W22H mutant, both kcat and kcat/Km(dihydrofolate) are substantially lower than the corresponding wild-type values. While both mutations weaken dihydrofolate binding, cofactor NADPH binding is not significantly altered. Fitting of the kinetic pH profiles to a general protonation scheme suggests that the proton affinity of dihydrofolate may be enhanced upon binding to the enzyme. We suggest that the function of tryptophan-22 may be to properly position the side chain of methionine-20 with respect to N5 of the substrate dihydrofolate.     

Kinetics of bilirubin oxidation catalysed by bilirubin oxidase in a water-in-oil microemulsion system

1. Bilirubin oxidase can catalyse the oxidation of its primary substrate, bilirubin, in a water-in-oil microemulsion, which consists of discrete nanometer-diameter water droplets dispersed in a continuous water-immiscible oil medium. The droplets are stabilized by a monolayer of the surfactant, cetyltrimethylammonium bromide present at the oil/water interface. 2. Spectroscopic evidence is presented to show that bilirubin solubilized in this system is located mainly in the surfactant layer, in a form accessible to the enzyme molecule. 3. Studies are presented on the enzyme-catalysed rate of bilirubin oxidation in this system, as a function of temperature, pH, water content, and substrate and enzyme concentrations. 4. The main conclusions are that the enzyme can efficiently oxidise bilirubin in microemulsions of low water content. The reaction obeys Michaelis-Menten kinetics. The optimal pH for the catalysis is 8.0. The efficiency of catalysis decreases sharply as the water content increases.     

A kinetic analysis of the interaction of human myeloperoxidase with hydrogen peroxide, chloride ions, and protons

The effect of H2O2, Cl-, and pH on human myeloperoxidase activity has been examined. The Km for H2O2 is shown to be affected by the combined presence of Cl- and acid pH conditions. The Km for H2O2 is independent of pH in the absence of Cl- and dependent on pH in the presence of Cl-. Conversely, the dependence of the Km for H2O2 on Cl- concentration increases as the pH decreases. A model is proposed in which Cl- has a dual role, acting both as a substrate and as an inhibitor. According to this model, the inhibitor Cl- binding site must be protonated prior to the binding of Cl- and is distinct from the substrate Cl- binding site which is unaffected by pH. The rate equation derived from this model is used to further analyze the data presented. The values of Km for H2O2 predicted by the rate equation are in good agreement with the experimentally determined values.     

Estimation of deuteration levels in whole cells and cellular proteins by 1H n.m.r. spectroscopy and neutron scattering.

For bovine erythrocyte acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7), the Michaelis parameters Vmax., and Km for the natural substrate acetylcholine were estimated as a function of pH and sodium chloride concentration by the pH-stat method. A single dissociation constant for Na+ binding (K = 7 X 10(-3) M) suffices to explain the salt dependence of Vmax./Km and of Km as well as the pH dependence of Vmax./Km and Vmax., Km being pH independent. This finding provides evidence for a specific effect of Na+, presumably by binding at the anionic subsite of the active centre. Na+ binding causes a 50-fold decrease in kcat./Km as well as a decrease of one unit in the pKa of both kcat./Km and kcat.. The intrinsic pKa in the absence of salt at 25 degrees C is about 7.5. Comparison of the degree of fit of the data to the Debeye-Huckel equation, in accordance with an alternative general salt effect, as well as published data for sodium and potassium chlorides also favour a specific salt effect.     

Kinetic independence of the subunits of cytosolic glutathione transferase from the rat.

The steady-state kinetics of the dimeric glutathione transferases deviate from Michaelis-Menten kinetics, but have hyperbolic binding isotherms for substrates and products of the enzymic reaction. The possibility of subunit interactions during catalysis as an explanation for the rate behaviour was investigated by use of rat isoenzymes composed of subunits 1, 2, 3 and 4, which have distinct substrate specificities. The kinetic parameter kcat./Km was determined with 1-chloro-2,4-dinitrobenzene, 4-hydroxyalk-2-enals, ethacrynic acid and trans-4-phenylbut-3-en-2-one as electrophilic substrates for six isoenzymes: rat glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4 and 4-4. It was found that the kcat./Km values for the heterodimeric transferases 1-2 and 3-4 could be predicted from the kcat./Km values of the corresponding homodimers. Likewise, the initial velocities determined with transferases 3-3, 3-4 and 4-4 at different degrees of saturation with glutathione and 1-chloro-2,4-dinitrobenzene demonstrated that the kinetic properties of the subunits are additive. These results show that the subunits of glutathione transferase are kinetically independent.     

The steady-state rate equation for cytochrome c oxidase based on a minimal kinetic scheme

A minimal catalytic cycle for cytochrome c oxidase has been suggested, and the steady-state kinetic equation for this mechanism has been derived. This equation has been used to simulate experimental data for the pH dependence of the steady-state kinetic parameters, kcat and Km. In the simulations the rate constants for binding and dissociation of cytochrome c and for two internal electron-transfer steps have been allowed to vary, whereas fixed experimental values (for pH 7.4) have been used for the other rate constants. The results show that the dissociation of the product, ferricytochrome c, cannot be rate-limiting under all conditions, but that intramolecular electron-transfer steps also limit the rate. They also demonstrate that Km can differ considerably from the dissociation constant for the cytochrome c-oxidase complex. Published values for the rate constant for the dissociation of ferricytochrome c are too small to account for the steady-state rates. It is suggested that, at high concentrations, ferryocytochrome c transfers an electron to a cytochrome c molecule which remains bound to the oxidase. This can also explain the nonhyperbolic kinetics, which is observed at low substrate concentrations.     

Plasminogen activation by single-chain urokinase in functional isolation. A kinetic study

The kinetics of the activation of Glu- and Lys-plasminogen by single-chain urokinase (sc urokinase) derived from the transformed human kidney cell line TCL-598 have been studied and compared with two-chain urokinase (tc urokinase). Plasminogen activation was determined by the increase in fluorescence polarization of fluorescein-labeled aprotinin, a high affinity inhibitor of plasmin. This methodology allows plasmin generation by sc urokinase to be measured in functional isolation, with no interfering generation of tc urokinase, sc urokinase was found to activate plasminogen to plasmin with apparent Michaelis-Menten-type kinetics. The Km for Glu-plasminogen activation was 47.7 microM, with a catalytic constant of 2.91 min-1. Lys-plasminogen activation by sc urokinase was characterized by a Km of 11.7 microM and a kcat of 5.60 min-1. The Km values for the activation of Glu- and Lys-plasminogen by tc urokinase were found to be similar to those for activation by sc urokinase (36.8 and 9.0 microM, respectively), but the catalytic constants were higher at 36.0 and 118 min-1, respectively. Therefore, on the basis of the catalytic efficiency kcat/Km, sc urokinase seems to have 16-27-fold lower activity than tc urokinase. This activity of sc urokinase is in contrast to its lack of activity against a low molecular weight peptide substrate (less than 0.2% of the activity of sc urokinase). The activation of sc urokinase to tc urokinase by plasmin was also characterized (Km = 3.0 microM, kcat = 105 min-1). Using these data, it was possible to calculate the theoretical rate of plasminogen activation by sc urokinase in the absence of aprotinin, when tc urokinase is generated by the action of plasmin. The calculated rate was in good agreement with that determined experimentally using the chromogenic substrate D-Val-Leu-Lys-p-nitroanilide. These data demonstrate that sc urokinase has properties which distinguish it from conventional serine protease zymogens. The lack of activity against low molecular weight peptide substrates demonstrates the inaccessibility of the substrate-binding pocket. However, there is a moderate activity against plasminogen, suggesting that plasminogen may be acting as both an effector and a substrate for sc urokinase.     

Subsite interactions of ribonuclease T1: viscosity effects indicate that the rate-limiting step of GpN transesterification depends on the nature of N

We report on the effect of the viscogenic agents glycerol and ficoll on the RNase T1 catalyzed turnover of GpA, GpC, GpU, and Torula yeast RNA. For wild-type enzyme, we find that the kcat/Km values for the transesterification of GpC and GpA as well as for the cleavage of RNA are inversely proportional to the relative viscosity of glycerol-containing buffers; no such effect is observed for the conversion of GpU to cGMP and U. The second-order rate constants for His40Ala and Glu46Ala RNase T1, two mutants with a drastically reduced kcat/km ratio, are independent of the microviscosity, indicating that glycerol does not affect the intrinsic kinetic parameters. Consistent with the notion that molecular diffusion rates are unaffected by polymeric viscogens, addition of ficoll has no effect on the kcat/Km for GpC transesterification by wild-type enzyme. The data indicate that the second-order rate constants for GpC, GpA, and Torula yeast RNA are at least partly limited by the diffusion-controlled association rate of substrate and active site; RNase T1 obeys Briggs-Haldane kinetics for these substrates (Km greater than Ks). Calculations suggest that the equilibrium dissociation constants (Ks) for the various GpN-wild-type enzyme complexes are virtually independent of N whereas the measured kcat values follow the order GpC greater than GpA greater than GpU. This is also revealed by the steady-state kinetic parameters of Tyr38Phe and His40Ala RNase T1, two mutants that follow simple Michaelis-Menten kinetics because of a dramatically reduced kcat value (i.e., Km = Ks).(ABSTRACT TRUNCATED AT 250 WORDS)     

Substituent effects on substrate activation and Michaelis-Menten Kinetic parameters in the alpha-chymotrypsin-catalyzed hydrolysis of phenyl acetates.

The effects of substituents on the steady state and pre-steady state kinetics in alpha-chymotrypsin [EC 3.4.21.1]-catalyzed hydrolysis were studied using substituted phenyl acetates. In the steady state hydrolysis, substrate activation, which had been observed and studied previously for p-nitrophenyl acetate, was also observed for p-bromo, p-chloro-, and m-methylphenyl acetates. Little activation was observed for p-acetyl-, m-nitro-, p-methyl-, and p-methoxyphenyl acetates. Addition of p-dichlorobenzene increased kcat for all substrates examined and greatly diminished the substrate activation for the activatable substrate(s) to activator binding site(s). The value of kcat decreased in accordance with increase of the sigma-value of substituents. On the other hand, kcat/Km (app) showed an opposite sigma- dependence, as was previously observed. In pre-steady state measurements, little burst was observed for more electron-donating substituents than m-nitro. The sigma dependence of kcat is apparently not consistent with the prediction derived from that of kcat/Km (app) on the basis of the usual two-step mechanism with a common acetyl-enzyme intermediate.     

Kinetics and sequence specificity of processing of prepilin by PilD, the type IV leader peptidase of Pseudomonas aeruginosa.

PilD, originally isolated as an essential component for the biogenesis of the type IV pili of Pseudomonas aeruginosa, is a unique endopeptidase responsible for processing the precursors of the P. aeruginosa pilin subunits. It is also required for the cleavage of the leader peptides from the Pdd proteins, which are essential components of an extracellular secretion pathway specific for the export of a number of P. aeruginosa hydrolytic enzymes and toxins. Substrates for PilD are initially synthesized with short, i.e., 6- to 8-amino-acid-long, leader peptides with a net basic charge and share a high degree of amino acid homology through the first 16 to 30 residues at the amino terminus. In addition, they all have a phenylalanine residue at the +1 site relative to the cleavage site, which is N methylated prior to assembly into the oligomeric structures. In this study, the kinetics of leader peptide cleavage from the precursor of the P. aeruginosa pilin subunit by PilD was determined in vitro. The rates of cleavage were compared for purified enzyme and substrate as well as for enzyme and substrate contained within total membranes extracted from P. aeruginosa strains overexpressing the cloned pilD or pilA genes. Optimal conditions were obtained only when both PilD and substrate were contained within total membranes. PilD catalysis of P. aeruginosa prepilin followed normal Michaelis-Menten kinetics, with a measured apparent Km of approximately 650 microM, and a kcat of 180 min-1. The kinetics of PilD processing of another type IV pilin precursor, that from Neisseria gonorrhoeae with a 7-amino-acid-long leader peptide, were essentially the same as that measured for wild-type P. aeruginosa prepilin. Quite different results were obtained for a number of prepilin substrates containing substitutions at the conserved phenylalanine at the +1 position relative to the cleavage site, which were previously shown to be well tolerated in vivo. Substitutions of methionine, serine, and cysteine for phenylalanine show that Km values remain close to that measured for wild-type substrate, while kcat and kcat/Km values were significantly decreased. This indicates that while the affinity of enzyme for substrate is relatively unaffected by the substitutions, the maximum rate of catalysis favors a phenylalanine at this position. Interesting, PilD cleavage of one mutated pillin (asparagine) resulted in a lower Km value of 52.5 microM, which indicates a higher affinity for the enzyme, as well as a lower kcat value of 6.1 min m(-1). This suggests that it may be feasible to design peptide inhibitors of PilD.     

The cyclic AMP-dependent protein kinase from bovine cardiac muscle is a homoserine kinase

The catalytic subunit of the cAMP-dependent protein kinase from bovine cardiac muscle phosphorylates homoserine in the synthetic peptide Leu-Arg-Arg-Ala-Hse-Leu-Gly. Phosphorylation of the primary alcohol of the homoserine residue was established via NMR spectroscopy. Two-dimensional correlated and nuclear Overhauser effect spectroscopies provided the sequence-specific chemical shift assignments of the substrate peptide and its phosphorylated counterpart. Coupled and decoupled 31P NMR experiments established the presence of phosphate on the homoserine residue. The maximal velocity (6.4 mumol/min.mg) obtained for homoserine-peptide phosphorylation at 12.5 mM Mg2+ compares favorably to the velocities observed for the corresponding serine- (21 mumol/min.mg), threonine- (3.2 mumol/min.mg), and hydroxyproline-peptides (1 mumol/min.mg). However, the Km for homoserine kinase activity is modest (1.3 mM) relative to the Km associated with the phosphorylation of the serine-containing substrate (22 microM). The effect of Mg2+ concentration on the kinetic parameters kcat, Km, and kcat/Km was investigated for both serine- and homoserine-peptides. Both substrates display similar kcat/Km versus [Mg2+] profiles, with the most notable difference that the optimal Mg2+ concentration is higher for the homoserine-containing peptide. In addition, the Km for the serine-peptide was found to be independent of [Mg2+], whereas the Km for the homoserine-peptide was observed to be dependent upon [Mg2+]. These results suggest that the long homoserine side chain may induce an unusually large off rate for the peptide and/or may misalign the hydroxyl moiety in the active site.     

Kinetic properties of triose-phosphate isomerase from Trypanosoma brucei brucei. A comparison with the rabbit muscle and yeast enzymes

The kinetic properties of Trypanosoma brucei brucei triose-phosphate isomerase are compared with those of the commercially available rabbit muscle and yeast enzymes and with published data on the chicken muscle enzyme. With glyceraldehyde 3-phosphate as substrate Km = 0.25 +/- 0.05 mM and kcat = 3.7 X 10(5) min-1. With dihydroxyacetone phosphate as substrate Km = 1.2 +/- 0.1 mM and kcat = 6.5 X 10(4) min-1. The pH dependence of Km and Vmax at 0.1 M ionic strength is in agreement with the results published for the yeast and chicken muscle enzymes. At ionic strength below 0.05 M the effect of a charged group specific for the trypanosomal enzyme and absent from the yeast and rabbit muscle enzymes becomes detectable. This effect significantly increases Km whereas Vmax becomes slightly higher. Trypanosomal triose-phosphate isomerase is inhibited by sulphate, phosphate and arsenate ions, by 2-phosphoglycolate and a number of documented inhibitors in the same concentration range as are the other triose-phosphate isomerases. The trypanocidal drug, Suramin inhibits T. brucei and rabbit muscle triose-phosphate isomerase to the same extent while leaving the yeast enzyme relatively unaffected.     

Energetics of proline racemase: racemization of unlabeled proline in the unsaturated, saturated, and oversaturated regimes

The interconversion of L- and D-proline catalyzed by proline racemase has been studied. The entire time course of the approach to equilibrium has been followed. After a short time the product concentration is significant, and the reaction runs under reversible conditions. As the total substrate concentration is increased, the system moves from the unsaturated regime into the saturated regime. At very high substrate levels under the reversible conditions used, the rate constant for substrate racemization falls, as the system moves into the "oversaturated" regime. Here, the net rate of the enzyme-catalyzed reaction is limited by the rate of return of the free enzyme from the form that liberates product back to the form that binds substrate. The results are analyzed in terms of the simple mechanism (table; see text) and illustrate the additional information that is available from reactions studied under reversible conditions. In the unsaturated region the value of the second-order rate constant kU (equivalent to kcat/Km) is 9 X 10(5) M-1 s-1 in each direction. In the saturated region, kcat = kcat = 2600 s-1 and Km = 2.9 mM. In the oversaturated region, the rate constant kO is 81 M s-1. The substrate concentration at which unsaturated and saturated terms contribute equally is 2.9 mM, and the substrate concentration at which saturated and oversaturated terms contribute equally is 125 mM.     

Diffusion-dependent rates for the hydrolysis reaction catalyzed by glyoxalase II from rat erythrocytes

Glyoxalase II from rat erythrocytes is a near optimal catalyst for the hydrolysis of S-D-lactoylglutathione in the sense that the magnitude of kcat/Km is limited, in large part, by the rate constant for diffusion-controlled encounter between substrate and active site. The experimental basis for this conclusion is derived from the dependencies of the kinetic properties of the enzyme on solution viscosity (pH 7, Ic = 0.1 M, 25 degrees C). When sucrose is used as a viscogenic agent, kcat/Km for S-D-lactoylglutathione (8.8 x 10(5) M-1 s-1) decreases markedly with increasing solution viscosity. This effect appears not to be due to a sucrose-induced change in the intrinsic kinetic properties of the enzyme, since kcat/Km for the slow substrate S-acetylglutathione (3.7 x 10(4) M-1 s-1) is nearly independent of solution viscosity. Quantitative treatment of the data using Stoke's law indicates that the rate of hydrolysis of S-D-lactoylglutathione will be approximately 50% diffusion limited when [substrate] much less than Km; the encounter complex between enzyme and substrate partitions nearly equally between product formation and dissociation to form free enzyme and substrate. The same conclusion is reached when glycerol is used as a viscogenic agent, once the apparent activation effect of glycerol on the intrinsic activity of the enzyme is taken into account. Finally, the rate of formation of the encounter complex between substrate and active site may be governed to a significant extent by charge-charge interactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure-Activity Relationship of a U-Type Antimicrobial Microemulsion System

The structure-activity relationship of a U-type antimicrobial microemulsion system containing glycerol monolaurate and ethanol at a 1∶1 mass ratio as oil phase and Tween 20 as surfactant were investigated along a water dilution line at a ratio of 80∶20 mass% surfactant/oil phase, based on a pseudo-ternary phase diagram. The differential scanning calorimetry results showed that in the region of up to 33% water, all water molecules are confined to the hydrophilic core of the reverse micelles, leading to the formation of w/o microemulsion. As the water content increases, the water gains mobility, and transforms into bicontinuous in the region of 33–39% water, and finally the microemulsion become o/w in the region of above 39% water. The microstructure characterization was confirmed by the dynamic light scattering measurements and freeze-fracture transmission electron microscope observation. The antimicrobial activity assay using kinetics of killing analysis demonstrated that the microemulsions in w/o regions exhibited relatively high antimicrobial activity against Escherichia coli and Staphylococcus aureus due to the antimicrobial oil phase as the continuous phase, while the antimicrobial activity started to decrease when the microemulsions entered the bicontinuous region, and decreased rapidly as the water content increased in the o/w region, as a result of the dilution of antimicrobial oil droplets in the aqueous continuous phase.     

Steady-state kinetics of combined oxidation of hydroquinone and o-dianisidine by hydrogen peroxide in the presence of horseradish peroxidase

The steady-state kinetics of horseradish peroxidase-catalyzed oxidation of hydroquinone was studied. Hydroquinone was shown to be a rapidly oxidizable substrate of the peroxidase. Values of kcat and Km for this substrate were determined in the pH range 4-7. The oxidation of hydroquinone and o-dianisidine was distinguished when both were present in the reaction mixture. o-Dianisidine was not oxidized until hydroquinone was completely converted. The rate of hydroquinone oxidation by peroxidase in the presence of o-dianisidine was 3-10 times higher than the rate of its individual oxidation. The activator decreased the Km for hydroquinone oxidation.     

The physical significance of Km in the prothrombinase reaction

Key kinetic parameters for the prothrombinase complex formed on membranes of phosphatidylserine (PS)/phosphatidylcholine (PC) (40/60) (Km = 0.12 microM, kcat = 11 s-1) or PS/PC (2/98) (Km = 0.40 microM, kcat = 11 s-1) differed only slightly. In contrast, the density of proteins on the membrane surface at the km differed greatly for the two membranes. The kinetics appeared unaffected by conditions where the number of phospholipid vesicles (2% PS) exceeded the number of protein molecules. These results establish that the Km for the prothrombinase reaction is determined by the concentration of prothrombin in solution rather than its density at the membrane surface. This system can be treated as a dissociable enzyme acting on a soluble substrate.     

Nature of rate-limiting steps in a compartmentalized enzyme system. Quantitation of dopamine transport and hydroxylation rates in resealed chromaffin granule ghosts

Using isolated chromaffin granule ghosts from bovine adrenal medullae, we have studied the kinetics of dopamine beta-monooxygenase (D beta M) activity as it is linked to dopamine transport. Measurements of the initial rates of transport and of transport-linked norepinephrine formation suggested that enzyme activity may be partially rate-limiting in the coupled carrier/enzyme system. This was confirmed by (i) measurements of initial rates of norepinephrine formation using deuterated substrate, which gave isotope effects greater than 2.0, and (ii) kinetic measurements using ghosts pulsed with varying concentrations of labeled dopamine, which indicated substantial substrate accumulation in the vesicle interior as a function of time. Initial rates of product formation, when combined with approximations of internal substrate concentrations, allowed estimates of Kcat and Km for intravesicular D beta M. Activation by external reductant was apparent in both initial rate parameters and the measurements of transients. Under conditions of optimal D beta M activity, the enzyme rate parameters (kcat = 0.31 nmol/s.mg and Km = 2 mM) indicated partial rate limitation compared to dopamine transport (kcat = 0.38 nmol/s.mg and Km = 32 microM). Compartmental analysis of the time curves, performed using numerical nonlinear least squares methods, gave least squares estimates of rate constants for a simple carrier mechanism and kcat values for D beta M which were consistent with estimates from initial rates.