A conformational change in phosphoglycerate dehydrogenase induced by a shift in pH.

The fluorescence of NADH bound to phosphoglycerate dehydrogenase (3-phosphoglycerate: NAD+ oxidoreductase, EC 1.1.1.95) decreased by 42% between pH 8.5 and 7.0 Serine, an allosteric inhibitor, quenched the fluorescence of enzyme-bound NADH by 29% at pH 8.5, but not at all at pH 7.0. The kinetics of the fluorescence change which occurred when the pH of an enzyme-NADH solution was rapidly shifted from 8.5 to 7.0 was measured using stopped-flow fluorimetry. The kinetics were first order, with a rate constant of 2.83 s-1. This rate constant was similar in magnitude to the rate constants for fluorescence quenching at pH 8.5 by saturating concentrations of serine and glycine, another allosteric inhibitor (Dubrow, R. and Pizer, L.I. (1977) J. Biol. Chem. 252, 1527-1538). These results indicate that the conformation of phosphoglycerate dehydrogenase at pH 7.0 is similar to, but not identical with, the serine-induced conformation at pH 8.5.     

Reduced S-adenosylhomocysteine hydrolase. Kinetics and thermodynamics for binding of 3'-ketoadenosine, adenosine, and adenine.

S-Adenosylhomocysteine hydrolase (SAHase) was resolved into apoenzyme and NAD+ by acidic ammonium sulfate treatment. The apoenzyme was catalytically inactive, but could be reconstituted to active enzyme with NAD+. Reduced SAHase (ENADH) that was prepared by reconstitution of the apoenzyme with NADH was catalytically inactive. ENADH was oxidized by 3'-ketoadenosine to active SAHase. The recovery of activity paralleled the oxidation of enzyme-bound NADH. The association rate constant for ENADH and 3'-ketoadenosine was 6.1 x 10(2) M-1 s-1, and the dissociation rate constant was calculated to be 4 x 10(-7) s-1. This association rate constant was considerably smaller than the association rate constant for adenosine and SAHase (greater than 10(7) M-1 s-1). However, the observed pseudo first-order rate constant for reaction of 3'-ketoadenosine with ENADH (0.6 s-1 with 1 mM 3'-ketoadenosine) approached kcat for the hydrolytic reaction (1.2 s-1). Thus, bound 3'-ketoadenosine probably reacted sufficiently rapidly with ENADH to be considered a kinetically competent intermediate. The dissociation constants of SAHase for adenosine and 4',5'-dehydroadenosine, substrates for the enzyme, were 9 and 14 microM, respectively. In contrast, the dissociation constants of ENADH for 3'-ketoadenosine and 4',5'-dehydro-3'-ketoadenosine, intermediates of the catalytic reaction, were significantly lower with values of 600 and 300 pM, respectively. The equilibrium constant for reduction of enzyme-bound NAD+ in the absence of an adenosine analogue, as estimated from cyanide binding studies, was 10-fold more favorable than that for free NAD+. ENADH was highly fluorescent (emission maximum 428 nm, excitation 340 nm) with a quantum yield that was six times that of free NADH. Since SAHase reduced by adenosine was not highly fluorescent, enzyme-bound intermediates quenched the fluorescence of enzyme-bound NADH. Adenosine and adenine quenched the fluorescence of ENADH. Cyanide formed a complex with SAHase that was analogous to ENADH. Adenine stabilized this complex sufficiently that addition of 65 microM adenine and 25 mM cyanide to SAHase caused total complex formation with loss of over 95% of the catalytic activity.     

Steady-state and pre-steady kinetic studies on mitochondrial sheep liver aldehyde dehydrogenase. A comparison with the cytoplasmic enzyme.

Kinetic studies were carried out on mitochondrial aldehyde dehydrogenase (EC 1.2.1.3) isolated from sheep liver. Steady-state studies over a wide range of acetaldehyde concentrations gave a non-linear double-reciprocal plot. The dissociation of NADH from the enzyme was a biphasic process with decay constants 0.6s-1 and 0.09s-1. Pre-steady-state kinetic data with propionaldehyde as substrate could be fitted by using the same burst rate constant (12 +/- 3s-1) over a wide range of propionaldehyde concentrations. The quenching of protein fluorescence on the binding of NAD+ to the enzyme was used to estimate apparent rate constants for binding (2 X 10(4) litre.mol-1.s-1) and dissociation (4s-1). The kinetic properties of the mitochondrial enzyme, compared with those reported for the cytoplasmic aldehyde dehydrogenase from sheep liver, show significant differences, which may be important in the oxidation of aldehydes in vivo.     

Steady-state and pre-steady-state kinetics of propionaldehyde oxidation by sheep liver cytosolic aldehyde dehydrogenase at pH 5.2. Evidence that the release of NADH remains rate-limiting in the enzyme mechanism at acid pH values

The kcat value for the oxidation of propionaldehyde by sheep liver cytosolic aldehyde dehydrogenase increased 3-fold, from 0.16 s-1 at pH 7.6 to 0.49 s-1 at pH 5.2, in parallel with the increase in the rate of displacement of NADH from binary enzyme.NADH complexes. A burst in nucleotide fluorescence was observed at all pH values consistent with the rate of isomerization of binary enzyme.NADH complexes constituting the rate-limiting step in the steady state. No substrate activation by propionaldehyde was observed at pH 5.2, but the enzyme exhibited dissociation/association behavior. The inactive dissociated form of the enzyme was favored by low enzyme concentration, low pH, and low ionic strength. Propionaldehyde protected the enzyme against dissociation.     

Elucidation of a [4Fe-4S] cluster degradation pathway: rapid kinetic studies of the degradation of Chromatium vinosum HiPIP

Irreversible disassembly of the 4Fe-4S cluster in Chromatium vinosum high-potential iron protein (HiPIP) has been investigated in the presence of a low concentration of guanidinium hydrochloride. From the dependence of degradation rate on [H+], it is deduced that at least three protons are required to trigger efficient cluster degradation. Under these conditions the protonated cluster shows broadened M?ssbauer signals, but delta EQ (1.1 mm/s) and delta (0.44 mm/s) are similar to the native form. Collapse of the protonated transition state complex, revealed by rapid-quench M?ssbauer experiments, occurs with a measured rate constant kobs approximately 0.72 +/- 0.35 s-1 that is consistent with results from time-resolved electronic absorption and fluorescence (kobs approximately 0.4 +/- 0.1 s-1) and EPR (kobs approximately 0.62 +/- 0.18 s-1) measurements. Apparently, guanidinium hydrochloride serves to perturb the tertiary structure of the protein, facilitating protonation of the cluster, but not degradation per se. Release of iron ions occurs even more slowly with kobs approximately 0.07 +/- 0.02 s-1, as determined by the appearance of the g = 4.3 EPR signal. Proton-mediated cluster degradation is sensitive to the oxidation state of the cluster, with the oxidized state showing a two-fold slower rate in acidic solutions as a result of increased electrostatic repulsion with the cluster. Consistent results are obtained from absorption, fluorescence, M?ssbauer and EPR measurements.     

Purine nucleoside phosphorylase. Kinetic mechanism of the enzyme from calf spleen.

Ribose 1-phosphate, phosphate, and acyclovir diphosphate quenched the fluorescence of purine nucleoside phosphorylase at pH 7.1 and 25 degrees C. The fluorescence of enzyme-bound guanine was similar to that of anionic guanine in ethanol. Guanine and ribose 1-phosphate bound to free enzyme, whereas inosine and guanosine were not bound to free enzyme in the absence of phosphate. Thus, synthesis proceeded by a random mechanism, and phosphorolysis proceeded by an ordered mechanism. Steady-state kinetic data for the phosphorolysis of 100 microM guanosine were fitted to a bifunctional kinetic model with catalytic rate constants of 22 and 1.3 s-1. The dissociation rate constants for guanine from the enzyme-guanine complex at high and low phosphate concentrations were similar to the catalytic rate constants. Fluorescence changes of the enzyme during phosphorolysis suggested that ribose 1-phosphate dissociated from the enzyme ribose 1-phosphate-guanine complex rapidly and that guanine dissociated from the enzyme-guanine complex slowly. The association and dissociation rate constants for acyclovir diphosphate, a potent inhibitor of the enzyme (Tuttle, J. V., and Krenitsky, T. A. (1984) J. Biol. Chem. 259, 4065-4069), were also dependent on phosphate concentration. The effects of phosphate are discussed in terms of a dual functional binding site for phosphate.     

NADH binding to porcine mitochondrial malate dehydrogenase.

The binding of NADH to porcine mitochondrial malate dehydrogenase in phosphate buffer at pH 7.5 has been studied by equilibrium and kinetic methods. Hyperbolic binding was obtained by fluorimetric titration of enzyme with NADH, in the presence or absence of hydroxymalonate. Identical results were obtained for titrations of NADH with enzyme in the presence or absence of hydroxymalonate, measured either by fluorescence emission intensity or by the product of intensity and anisotropy. The equilibrium constant for NADH dissociation was 3.8 +/- 0.2 micrometers, over a 23-fold range of enzyme concentration, and the value in the presence of saturating hydroxymalonate was 0.33 +/- 0.02 micrometer over a 10-fold range of enzyme concentration. The rate constant for NADH binding to the enzyme in the presence of hydroxymalonate was 3.6 X 10(7) M-1 s-1, while the value for dissociation from the ternary complex was 30 +/- 1 s-1. No limiting binding rate was obtained at pseudo-first order rate constants as high as 200 s-1, and the rate curve for dissociation was a single exponential for at least 98% of the amplitude. In addition to demonstrating that the binding sites are independent and indistinguishable, the absence of effects of enzyme concentration on the KD value indicates that NADH binds with equal affinity to monomeric and dimeric enzyme forms.     

Elementary steps in the reaction mechanism of chicken liver fatty acid synthase. Acylation of specific binding sites

The mechanism of acyl enzyme formation from acyl-CoA derivatives was studied for chicken liver fatty acid synthase in 0.1 M potassium phosphate (pH 7.0) and 1 mM EDTA at 23 degrees C. Three mechanistically important acyl-binding sites exist: a cysteine, 4'-phosphopantetheine, and a hydroxyl (serine). The cysteine was specifically labeled with iodoacetamide, and chemical modification of this labeled enzyme with chloroacetyl-CoA resulted in additional covalent labeling of 4'-phosphopantetheine. Reaction of the enzyme with acetyl-CoA results in 47% oxyester formation, whereas with malonyl-CoA and butyryl-CoA, 57 and 80% are oxyesters, respectively, as judged by treatment of the denatured enzyme with hydroxylamine. Limited proteolysis with trypsin followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the reactive hydroxyl and cysteine are on the same peptide. Butyryl-CoA is a relatively poor primer for steady state fatty acid synthesis, probably because transfer from the hydroxyl-binding site to 4'-phosphopantetheine is inefficient. Quenched flow studies indicate that the rate constants for transfer of acetyl from enzyme-bound acetyl-CoA to native, iodoacetamide-labeled, and iodoacetamide-chloroacetyl-labeled enzyme are 43, 110, and 150 s-1. These results can be interpreted in terms of a random acylation of the hydroxyl, 4'-phosphopantetheine, and cysteine by enzyme-bound acetyl-CoA with rate constants of 150 s-1, less than 110 s-1, and less than 43 s-1, respectively. Alternatively the latter two rate constants could be characteristic of intramolecular transfer between enzyme acylation sites. Structural constraints apparently prevent all three acylation sites from being occupied simultaneously. The rate of deacetylation of the acetylated enzyme by enzyme-bound CoA also is most rapid for the iodoacetamide-chloroacetyl-labeled enzyme.     

Transient kinetics of heparin-catalyzed protease inactivation by antithrombin III. Characterization of assembly, product formation, and heparin dissociation steps in the factor Xa reaction

The kinetics of alpha-factor Xa inhibition by antithrombin III (AT) were studied in the absence and presence of heparin (H) with high affinity for antithrombin by stopped-flow fluorometry at I 0.3, pH 7.4 and 25 degrees C, using the fluorescence probe p-aminobenzamidine (P) and intrinsic protein fluorescence to monitor the reactions. Active site binding of p-aminobenzamidine to factor Xa was characterized by a 200-fold enhancement and 4-nm blue shift of the probe fluorescence emission spectrum (lambda max 372 nm), 29-nm red shift of the excitation spectrum (lambda max 322 nm), and dissociation constant (KD) of about 80 microM. Under pseudo-first order conditions [( AT]0, [H]0, [P]0 much greater than [Xa]0), the observed factor Xa inactivation rate constant (kobs) measured by p-aminobenzamidine displacement or residual enzymatic activity increased linearly with the "effective" antithrombin concentration (i.e. corrected for probe competition) up to 300 microM in the absence of heparin, indicating a simple bimolecular process with a rate constant of 2.1 x 10(3) M-1 s-1. In the presence of heparin, a similar linear dependence of kobs on effective AT.H complex concentration was found up to 25 microM whether the reaction was followed by probe displacement or the quenching of AT.H complex protein fluorescence due to heparin dissociation, consistent with a bimolecular reaction between AT.H complex and free factor Xa with a 300-fold enhanced rate constant of 7 x 10(5) M-1 s-1. Above 25 microM AT.H complex, an increasing dead time displacement of p-aminobenzamidine and a downward deviation of kobs from the initial linear dependence on AT.H complex concentration were found, reflecting the saturation of an intermediate Xa.AT.H complex with a KD of 200 microM and a limiting rate of Xa-AT product complex formation of 140 s-1. Kinetic studies at catalytic heparin concentrations yielded a kcat/Km for factor Xa at saturating antithrombin of 7 x 10(5) M-1 s-1 in agreement with the bimolecular rate constant obtained in single heparin turnover experiments. These results demonstrate that 1) the accelerating effect of heparin on the AT/Xa reaction is at least partly due to heparin promoting the ordered assembly of antithrombin and factor Xa in an intermediate ternary complex and that 2) heparin catalytic turnover is limited by the rate of conversion of the ternary complex intermediate to the product Xa-AT complex with heparin dissociation occurring either concomitant with this step or in a subsequent faster step.     

NADH fluorescence lifetime analysis of the effect of magnesium ions on ALDH2

Aldehyde dehydrogenase 2 (ALDH2) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg(2+) ions influence ALDH2 activity in part by increasing NADH binding affinity. Traditional fluorescence measurements monitor the blue shift of the NADH fluorescence spectrum to study ALDH2-NADH interactions. By using time-resolved fluorescence spectroscopy, we have resolved the fluorescent lifetimes (τ) of free NADH (τ=0.4 ns) and bound NADH (τ=6.0 ns). We used this technique to investigate the effects of Mg(2+) on the ALDH2-NADH binding characteristics and enzyme catalysis. From the resolved free and bound NADH fluorescence signatures, the K(D) for NADH with ALDH2 ranged from 468 μM to 12 μM for Mg(2+) ion concentrations of 20 to 6000 μM, respectively. The rate constant for dissociation of the enzyme-NADH complex ranged from 0.4s(-1) (6000 μM Mg(2+)) to 8.3s(-1) (0 μM Mg(2+)) as determined by addition of excess NAD(+) to prevent re-association of NADH and resolving the real-time NADH fluorescence signal. The apparent NADH association/re-association rate constants were approximately 0.04 μM(-1)s(-1) over the entire Mg(2+) ion concentration range and demonstrate that Mg(2+) ions slow the release of NADH from the enzyme rather than promoting its re-association. We applied NADH fluorescence lifetime analysis to the study of NADH binding during enzyme catalysis. Our fluorescence lifetime analysis confirmed complex behavior of the enzyme activity as a function of Mg(2+) concentration. Importantly, we observed no pre-steady state burst of NADH formation. Furthermore, we observed distinct fluorescence signatures from multiple ALDH2-NADH complexes corresponding to free NADH, enzyme-bound NADH, and, potentially, an abortive NADH-enzyme-propanal complex (τ=11.2 ns).     

Calcium release from calmodulin and its C-terminal or N-terminal halves in the presence of the calmodulin antagonists phenoxybenzamine and melittin measured by stopped-flow fluorescence with Quin 2 and intrinsic tyrosine. Inhibition of calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum

Calcium dissociation from the C-terminal and N-terminal halves of calmodulin, intact bovine brain calmodulin and the respective phenoxybenzamine complexes or melittin complexes was measured directly by stopped-flow fluorescence with the calcium chelator Quin 2 and, when possible, also by protein fluorescence using endogenous tyrosine fluorescence by mixing with EGTA. Calcium dissociation from the C-terminal half of calmodulin, which contains only the two high-affinity calcium-binding sites, and from intact calmodulin was monophasic, with good correlation of the rates of calcium dissociation obtained by the two methods. The apparent rates with Quin 2 and endogenous tyrosine fluorescence were 13.4 s-1 and 12.8 s-1, respectively, in the C-terminal half and 10.5 s-1 and 10.8 s-1, respectively, in intact calmodulin (pH 7.0, 25 degrees C, 100 mM KCl). Alkylation of the C-terminal half resulted in a biphasic calcium dissociation (Quin 2: kobs 1.90 s-1 and 0.73 s-1 respectively; tyrosine: kobs 1.65 s-1 and 0.61 s-1 respectively). Alkylation of intact calmodulin resulted in a four-phase calcium dissociation measured with Quin 2 (kobs 85.3 s-1, 11.1 s-1, 1.92 s-1 and 0.59 s-1); the latter two phases are assumed to represent calcium release from high-affinity sites since they correspond to the biphasic tyrosine fluorescence change in intact alkylated calmodulin (kobs 2.04 s-1 and 0.53 s-1 respectively) and the rate parameters determined in the C-terminal half. Evidently perturbation of the calcium-binding sites by alkylation reduces the rate of calcium dissociation and allows a distinction to be made between dissociation from each of the two high-affinity sites as well as the distinct conformational change on dissociation of each calcium. Alkylation of the N-terminal half resulted in biphasic calcium release with rates (kobs 153 s-1 and 10.9 s-1 respectively) similar to those observed in intact alkylated calmodulin. The rates of calcium dissociation from calmodulin-melittin or fragment-melittin complexes, measured with Quin 2, were slower and monophasic in the C-terminal half (kobs 1.12 s-1), biphasic in the N-terminal half (kobs 140 s-1 and 26.8 s-1 respectively) and triphasic in intact calmodulin (kobs 126 s-1, 12.1 s-1 and 1.38 s-1). Calmodulin antagonists thus increase the apparent calcium affinity of high and low-affinity sites mainly due to a reduced calcium 'off rate', presumably because of conformation restrictions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetic mechanism of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase

IMP dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with conversion of NAD+ to NADH. This reaction is the rate-limiting step in de novo guanine nucleotide biosynthesis. IMPDH is a target for antitumor, antiviral, and immunosuppressive chemotherapy. We have determined the complete kinetic mechanism for IMPDH from Tritrichomonas foetus using ligand binding, isotope effect, pre-steady-state kinetic, and rapid quench kinetic experiments. Both substrates bind to the free enzyme, which suggests a random mechanism. IMP binds to the enzyme in two steps. Two steps are also involved when IMP binds to a mutant IMPDH in which the active site Cys is substituted with a Ser. This observation suggests that this second step may be a conformational change of the enzyme. No Vm isotope effect is observed when [2-2H]IMP is the substrate which indicates that hydride transfer is not rate-limiting. This result is confirmed by the observation of a pre-steady-state burst of NADH production when monitored by absorbance. However, when NADH production was monitored by fluorescence, the rate constant for the exponential phase is 5-10-fold lower than when measured by absorbance. This observation suggests that the fluorescence of enzyme-bound NADH is quenched and that this transient represents NADH release from the enzyme. The time-dependent formation and decay of [14C]E-XMP intermediates was monitored using rapid quench kinetics. These experiments indicate that both NADH release and E-XMP hydrolysis are rate-limiting and suggest that NADH release precedes hydrolysis of E-XMP.     

Serine activation is the rate limiting step of tRNASer aminoacylation by yeast seryl tRNA synthetase.

Using the quenched flow technique the mechanism of seryl tRNA synthetase action has been investigated with respect to the presteady state kinetics of individual steps. Under conditions where the strong binding sites of the enzyme are nearly saturated and the steady state turnover number is about 1 s-1, rate constants of four different processes have been determined: steps connected with substrate associations are relatively slow (12 s-1 for the entire process); activation of serine is the rate determining step (about 1.2 s-1 in presence of tRNASer); whereas the transfer of serine onto tRNASer (35 s-1) and the dissociation of seryl tRNASer (70 s-1) are fast. Similar kinetic parameter seem to hold also for the steady state reactions. This conclusion is based on a detailed study of the substrate, product, and Mg2+ concentration dependence of the transfer reaction. The results also indicate that a second serine binding site is operative. Since the transfer of serine from a preformed adenylate complex onto tRNASer is fast, seryl adenylate seems to be a kinetically competent intermediate of the aminoacylation reaction although, of course, alternative mechanisms cannot be excluded.     

A direct fluorescence study of the transient steps induced by calcium binding to sarcoplasmic reticulum ATPase

The sarcoplasmic reticulum intrinsic fluorescence level was closely correlated with the ATPase functional state, from pH 5.5 to 8.5. The fluorescence signal was used in stopped flow measurements for direct study of transient pump kinetics after calcium binding or removal. The signal change time course, which depends solely on the free calcium concentration in the observation chamber, was analyzed as a single exponential. Rate constants (kobs) were relatively slow (5 to 20 s-1), indicating multistep interaction between calcium and the transport protein. At pH 7 and 20 degrees C, and in the presence of 100 mM potassium and 1 to 20 mM MgCl2, kobs first decreased, and then increased as the calcium concentration rose. Similar experiments were performed at pH 6. Data were analyzed according to a scheme in which sarcoplasmic reticulum . calcium complex formation is controlled by a slow isomerization step occurring either before or after the rapid calcium binding to the high affinity site. The results are discussed with reference to published rapid quenching experiments. Under our conditions, i.e. in the absence of a calcium gradient across the membrane, the calcium pump cycle step in which reorientation of the calcium binding sites occurs cannot be identified with the isomerization step mentioned above.     

Structure elucidation of two differentiation inducing factors (DIF-2 and DIF-3) from the cellular slime mould Dictyostelium discoideum.

Acylation of the aldehyde dehydrogenase.NADH complex by acetic anhydride leads to the production of acetaldehyde and NAD+. By monitoring changes in nucleotide fluorescence, the rate constant for acylation of the active site of the *enzyme.NADH complex was found to be 11 +/- 3 s-1. The rate of acylation by acetic anhydride at the group that binds aldehydes on the oxidative pathway is clearly rapid enough to maintain significant steady-state concentrations of the required active-site-acylated *enzyme.NADH intermediate despite the rapid hydrolysis of this *enzyme.acyl.NADH intermediate (5-10 s-1) [Blackwell, Motion, MacGibbon, Hardman & Buckley (1987) Biochem. J. 242, 803-808]. Hence reversal of the normal oxidative pathway can occur. However, although acylation of the aldehyde dehydrogenase.NADH complex by 4-nitrophenyl acetate also occurs rapidly with a rate constant of 10.9 +/- 0.6 s-1, even under the most extreme trapping conditions only very small amounts of acetaldehyde are detected [Loomes & Kitson (1986) Biochem. J. 235, 617-619]. Furthermore enzyme-catalysed hydrolysis of 4-nitrophenyl acetate is limited by the rate of deacylation of a group on the enzyme (0.4 s-1), which is an order of magnitude less than deacylation of the group at the active site (5-10 s-1). It is concluded that the enzyme-catalysed 4-nitrophenyl ester hydrolysis involves a group on the enzyme that is different from the active-site group that binds aldehydes on the normal oxidative pathway.     

The kinetics of the reversible inhibition of heart lactate dehydrogenase through the formation of the enzyme–oxidized nicotinamide–adenine dinucleotide–pyruvate compound

The inhibition of lactate dehydrogenase at high pyruvate concentration was studied in three ways. First, a rapid decrease in the rate of the enzyme reaction was observed; secondly, the rate of formation of a pyruvate-NAD(+) compound was followed by the change in E(325); thirdly, the rate of quenching of the protein fluorescence was measured. The data obtained at pH6.0 at different temperatures and ionic strengths as functions of pyruvate, NAD(+) and enzyme concentrations show that the extent of inhibition can be correlated with the reversible formation of a compound between pyruvate and enzyme-bound NAD(+). It is suggested that the detailed kinetic analysis of the formation of this abortive ternary compound will give pertinent information about properties of the enzyme-NAD(+) compound involved in the normal catalytic process.     

Elementary steps in the reaction mechanism of chicken liver fatty acid synthase: reduced nicotinamide adenine dinucleotide phosphate binding and formation and reduction of acetoacetyl-enzyme

The kinetics of reduced nicotinamide adenine dinucleotide phosphate (NADPH) binding to fatty acid synthase from chicken liver and of the reduction of enzyme-bound acetoacetyl by NADPH (beta-ketoacyl reductase) and the steps leading to formation of the acetoacetyl-enzyme have been studied in 0.1 M potassium phosphate-1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.0, at 25 degrees C by monitoring changes in NADPH fluorescence with a stopped-flow apparatus. Improved fluorescence detection has permitted the use of NADPH concentrations as low as 20 nM. The kinetics of the binding of NADPH to the enzyme is consistent with a simple bimolecular binding mechanism and four equivalent sites on the enzyme (presumably two beta-ketoacyl reductase sites and two enoyl reductase sites). The bimolecular rate constant is 12.7 X 10(6) M-1 s-1, and the dissociation rate constant is 76.7 s-1, which gives an equilibrium dissociation constant of 6.0 microM. The formation of the acetoacetyl-enzyme and its subsequent reduction by NADPH could be analyzed as two consecutive pseudo-first-order reactions by mixing enzyme-NADPH with acetyl-CoA and malonyl-CoA under conditions where [acetyl-CoA], [malonyl-CoA] much greater than [enzyme] much greater than [NADPH]. From the dependence of the rate of reduction of aceto-acetyl-enzyme by NADPH on enzyme concentration, an independent estimate of the equilibrium dissociation constant for NADPH binding to the enzyme of 5.9 microM is obtained, and the rate constant for the reduction is 17.5 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Luminescence study of the conformation behavior of alcohol dehydrogenase from horse liver during substrate binding

The luminescence quenching and conformational behavior of alcohol dehydrogenase from horse liver upon substrate binding has been studied. It was shown that the binding of NADH and NAD+ to the enzyme resulted in the quenching of Trp-314 luminescence, whereas the luminescence of Trp-15 was not quenched. In this case non-radiating energy transfer from Trp-314 to NADH was observed. An essential energy transfer from Trp-15 to NADH and between the two Trp-314 of both subunits of the enzyme was not revealed. The quenching of the enzyme luminescence upon NAD+ binding was, mainly, caused by NAD+ reduction up to NADH. It was assumed, that the release of the proton upon NAD+ binding occurred due to the reduction. Binding of ethanol, ADP or adenosine did not result in essential conformational changes of the enzyme.     

Uni-site catalysis in thylakoids. The influence of membrane energization on ATP hydrolysis and ATP-Pi exchange

ATP-hydrolysis was measured with thylakoid membranes during continuous illumination. The concentrations of free and enzyme-bound ATP, ADP and Pi were measured using either cold ATP, [gamma-32P]ATP or [14C]ATP. The concentration of free ATP was constant, free ADP and enzyme-bound ATP were below the detection limit. Nevertheless, [gamma-32P]ATP was bound, hydrolyzed and 32Pi was released. The ADP was not released from the enzyme but cold Pi was bound from the medium, cold ATP was resynthesized and released. A quantitative analysis gave the following rate constants: ATP-binding kATP = 2 . 10(5) M-1 s-1, ADP-release: kADP less than 10(-2)s-1, Pi-release: kPi = 0.1 s-1. These rate constants are considerably smaller than under deenergized conditions. The rate constant for the release of ATP can be estimated to be at least 0.2 s-1 under energized conditions. Obviously, energization of the membrane, i.e. protonation of the enzyme leads mainly to a decrease of the rate of ATP-binding, to an increase of the rate of ATP release and to a decrease of the rate of ADP-release.     

Unusual transient- and steady-state kinetic behavior is predicted by the kinetic scheme operational for recombinant human dihydrofolate reductase

Association and dissociation rate constants obtained by stopped-flow spectroscopy have permitted definition of a kinetic scheme for recombinant human dihydrofolate reductase that correctly predicts full time course kinetics of the enzymatic reaction over a wide range of substrate and product concentrations. The scheme is complex compared with that for the bacterial enzyme and involves branched pathways. It successfully accounts for observed rapid hysteresis preceding steady state and for the nonhyperbolic dependence of steady-state rate on substrate and product concentrations. The major branch point in the catalytic cycle occurs at E.NADP.H4folate because either NADP or H4folate can dissociate from the ternary product complex (koff = 84 s-1 and 46 s-1, respectively). The rate of conversion of enzyme-bound substrates to products is very fast (k = 1360 s-1) and nearly unidirectional (Kequ = 37) so that other steps limit the catalytic rate. At saturating substrate concentrations these steps include release of NADP and H4folate from E.NADP.H4folate and release of products from the two abortive complexes E.NADPH.H4folate (koff = 225 s-1) and E.NADP.H4folate (koff = 4.6 s-1). Since NADP dissociates slowly from E.NADP.H2folate nearly 90% of the enzyme accumulates as this complex at steady state. Nonetheless, the catalytic rate is maintained at 12 s-1 by rapid flux of a small portion of the enzyme through an alternate branch. At physiological concentrations of substrates and products the steady-state rate is limited primarily by the rate of H2folate binding to E.NADPH so that the enzyme is extremely efficient.