Studies on the succinate dehydrogenating system. I. Kinetics of the succinate dehydrogenase interaction with a semiquindiimine radical of N,N,N',N'-tetramethyl-p-phenylenediamine.

1. The activities of the soluble reconstitutively active succinate dehydrogenase (EC 1.3.99.1) measured with three artificial electron acceptors, e.g. ferricyanide, phenazine methosulfate and free radical of N,N,N',N'-tetramethyl-p-phenylenediamine (WB), have been compared. The values estimated by extrapolation to infinite acceptor concentration using double reciprocal plots 1/v versus 1/[acceptor] are nearly the same for ferricyanide and phenazine methosulfate and about twice as high for the WB. 2. The double reciprocal plots 1/v versus 1/[succinate] in the presence of malonate at various concentrations of WB give a series of straight lines intercepting in the third quadrant. The data support the mechanism of the overall reaction, in which the reduced enzyme is oxidized by WB before dissociation of the enzyme-product complex. 3. The dependence of the rate of the overall reaction on WB concentration shows that only one kinetically significant redox site of the soluble succinate dehydrogenase is involved in the reduction of WB. 4. Studies of the change of V and Km values during aerobic inactivation of the soluble enzyme suggest that only 'the low Km ferricyanide reactive site' (Vinogradov, A.D., Gavrikova, E.V. and Goloveshkina, V.G. (1975) Biochem. Biophys, Res. Commun. 65, 1264--1269) is involved in reoxidation of the reduced enzyme by WB. 5. The pH dependence of V for the succinate-WB reductase reaction shows that the group of the enzyme with the pKa value of 6.7 at 22 degrees C is responsible for the reduction of dehydrogenase in the enzyme-substrate complex. 6. When WB interacts with the succinate-ubiquinone region of the respiratory chain, the double reciprocal plot 1/v versus 1/[WB] gives a straight line. The thenoyltrifluoroacetone inhibition of succinate-ubiquinone reductase or extraction of ubiquinone alter the 1/v versus 1/[WB] plots for the curves with a positive initial slope intercepting the ordinate at the same V as in the native particles. The data support the mechanism of succinate-ubiquinone reduction, in which no positive modulation of succinate dehydrogenase by ubiquinone exist in the membrane.     

Steady-state kinetic analysis for the reaction of ammonium and alkylammonium ions with methylamine dehydrogenase from bacterium W3A1

The steady-state kinetic mechanism for the reaction of n-alkylamines and phenazine ethosulfate (PES) or phenazine methosulfate (PMS) with methylamine dehydrogenase from bacterium W3A1 is found to be of the ping-pong type. This conclusion is based on the observations that 1/v versus 1/[methylamine] or 1/[butylamine] plots, at various constant concentrations of an oxidizing substrate, and 1/v versus 1/[PES] or 1/[PMS] plots, at various constant concentrations of a reducing substrate, are parallel. Additionally, the values of kcat/Km for four n-alkylamines are identical when PES is the oxidizing substrate, as were the kcat/Km values for four reoxidizing substrates when methylamine was the reducing substrate. Last, analysis of steady-state kinetic data obtained when methylamine and propylamine are presented to the enzyme simultaneously and PES and PMS are used simultaneously also supports the involvement of a ping-pong mechanism. The enzymic reaction with either methylamine or PES is dependent on the ionic strength, and the data indicate that each interacts with an anionic site on methylamine dehydrogenase. The presence of ammonium ion at low concentration activates the enzyme, but at high concentration this ion is a competitive inhibitor in the reaction involving methylamine and the enzyme. A complete steady-state mechanism describing these ammonia effects is presented and is discussed in light of the nature of the pyrroloquinoline quinone cofactor covalently bound to the enzyme.     

Kinetic studies on a novel sulfotransferase from Eubacterium A-44, a human intestinal bacterium

A novel sulfotransferase purified from a human intestinal bacterium stoichiometrically catalyzed the transfer of a sulfate group of phenylsulfate esters to phenolic compounds. Vmax values of the enzyme reaction were measured with various concentrations of a sulfate donor substrate, p-nitrophenylsulfate, and of a sulfate acceptor substrate, tyramine. Double reciprocal plots of the acceptor concentration and Vmax showed a linear correlation. One of the reaction products, tyramine O-sulfate, competitively inhibited the enzyme as to a donor substrate, p-nitrophenylsulfate (PNS), but the other reaction product, p-nitrophenol (PNP), noncompetitively inhibited it as to PNS. These kinetic data suggest that the sulfate transfer reaction proceeds according to a ping pong bi bi mechanism. The enzyme was activated by Mg2+ and inhibited by EDTA, which suggests that it is a metalloenzyme.     

Purification and some properties of the glycolipid transfer protein from pig brain

A glycolipid-specific lipid transfer protein has been purified to apparent homogeneity from pig brain post-mitochondrial supernatant. The purified protein was obtained after about 6,000-fold purification at a yield of 19%. Evidence for the homogeneity of the purified protein includes the following: (i) a single band in acidic gel electrophoresis, in sodium dodecyl sulfate-gel electrophoresis, (ii) a single band in analytical gel isoelectric focusing, (iii) exact correspondence between the glycolipid transfer activity and stained protein absorbance in the acidic gel electrophoresis, and (iv) coincidence between the transfer activity and protein absorption at 280 nm in gel filtration through Ultrogel AcA 54. The protein has an isoelectric point of about 8.3 and a molecular weight of 22,000, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A molecular weight of 15,000 was calculated from AcA 54 gel filtration. The amino acid composition has been determined. The protein binds [3H]galactosylceramide but not [3H]phosphatidylcholine. Under the conditions used, 1 mol of the transfer protein bound about 0.13 mol of [3H]galactosylceramide. The glycolipid transfer protein-[3H]galactosylceramide complex was isolated by a Sephadex G-75 chromatography. An incubation of the complex with liposomes resulted in the transfer of [3H]galactosylceramide from the complex to the acceptor liposomes. The result indicates that the complex functions as an intermediate in the glycolipid transfer reaction. The protein facilitates the transfer of [3H]galactosylceramide from donor liposomes to acceptor liposomes lacking in glycolipid as well as to acceptor liposomes containing galactosylceramide.     

Aminoacylaminonucleoside inhibitors of protein synthesis. A new approach for evaluating their potency

In a model system derived from Escherichia coli, Ac[3H]Phe-puromycin is produced in a pseudo-first-order reaction between the preformed Ac[3H]Phe-tRNA-poly(U)-ribosome complex (complex C) and excess puromycin [Kalpaxis et al. Eur. J. Biochem. 154, 267, 1986]. Amicetin and gougerotin inhibit this reaction to various degrees depending on whether or not complex C is allowed to interact with the inhibitor (I) prior to the addition of puromycin (S). The kinetic analysis shows a phase where competitive inhibition can be observed provided that S and I are added simultaneously. After preincubating C with I, the inhibition becomes of the mixed non-competitive type. The Ki (the dissociation constant of the CI complex), calculated from the competitive plot, is 20.0 microM for amicetin and 15.0 microM for gougerotin. This inhibition constant (Ki) cannot distinguish amicetin from gougerotin. Its acceptance as a criterion of potency does not explain why after preincubation amicetin proves to be a stronger inhibitor than gougerotin. The determination of the apparent catalytic rate constants of peptidyltransferase at various inhibitor concentrations and the appropriate replotting of these rate constants distinguish amicetin from gougerotin. A new approach for evaluating the potency of these inhibitors is proposed. The familiar Ki is supplemented with an apparent kinetic constant obtained from a replot in which the intercepts of the double-reciprocal plots (1/kobs versus 1/[S]) are plotted versus the inhibitor concentration.     

Molecular mechanism for the inhibition of acetylcholinesterase enzyme by organophosphorothionates

The different mechanisms, whereby EPN and malathion inhibit the action of cholinesterase on acetylcholine, are described. Partially purified brain enzyme was used for the kinetic studies. The approach of the theory of Krupka and Laidler was followed. The ratio of [S]I opt/[S]opt = 1 + Ki [I] to the first power was found with malathion but to the square root of (1 + Ki [I]) 1/2 with EPN. The intercept on the slope axis of plots of slopes of (1/V not equal to [I]) against the reciprocal of substrate concentrations showed a non-zero value in the case of EPN and a zero value in the case of malathion. Accordingly, and based on the above theory, it seems that malathion acts as a competitive inhibitor of cholinesterase while EPN seems to be a mixed type inhibitor.     

The effect of energization on the apparent Michaelis–Menten constant for oxyge in nmitochondrial respiration

Lineweaver-Burk plots of 1/v against 1/[O(2)] for rat liver mitochondrial respiration with succinate or ascorbate+NNN'N'-tetramethyl-p-phenylenediamine as substrates are non-linear. In state 3u (uncoupled by trifluoromethoxycarbonyl cyanide phenylhydrazone) such plots tend to be concave upward, whereas in state 4 (energized) the plots were concave downward. The apparent K(m) for oxygen is larger in state 4 than in state 3u, despite the higher turnover in the latter system. It is postulated that at least one reversible reaction occurs between cytochrome c and cytochrome c oxidase, whose rate is increased on energization (reversed electron transfer); a model including such a reaction is proposed which accounts semiquantitatively for the observations.     

Interaction of substrates and substrate-like inhibitors with the peptidyltransferase center from Escherichia coli ribosomes

The binding isotherms of CACCA(3'NHPhe----Ac) and CACCA(3'NHPhe) to E. coli ribosomes and 50S subunits were measured. A theoretical model of adsorption for the case of cooperative interaction between two ligands adsorbed on a ribosome was designated. The analysis of the experimental binding isoterms leads to the following conclusions. A ribosome (or subunit) binds one CACCA (3'NHPhe----Ac) molecule to donor site of the peptidyl transferase center, but two CACCA (3'NHPhe) molecules to both donor and acceptor sites. The binding of CACCA (3'NHPhe) to ribosomes (or subunits) is a cooperative process, characterized by the cooperativity coefficient tau = 40 +/- 5 or more. When model substrates CACCA-Phe, CACCA-Leu and CACCA-Val were taken instead of CACCA (3'NHPhe) in the incubation mixture with ribosomes, dipeptides were obtained even in the case, when ratio [model substrate]: [ribosome] (in moles) was much lower than 1. Puromycin binding to acceptor site with constant (1-2) X 10(4) M-1 also stimulates CACCA(3'NHPhe----Ac) adsorption to the donor site of ribosomes with cooperativity coefficient being equal to 1.5-2.5. It is also shown that cytidine 5'-phosphate binding to the donor site increases kappa cat of the reaction of minimal donors with CACCA-Phe by 1.5 orders of magnitude but has no effect on Km of this reaction. These facts point out that cytidine 5'-phosphate being adsorbed on the corresponding area of the donor site leads to the conversion of low-productive complex [ribosome + minimal donor substrate + acceptor substrate] into high-productive complex [ribosome + minimal donor substrate + acceptor substrate + cytidine 5'-phosphate].     

Kinetic model of the epidermal growth factor (EGF) receptor tyrosine kinase and a possible mechanism of its activation by EGF.

The tyrosine kinase activity of the epidermal growth factor receptor (EGFR-TK) was determined at varying poly-Glu6Ala3Tyr1 (GAT) or [Val5]-angiotensin II (AT) and constant ATP concentrations and vice versa. With GAT as substrate, double reciprocal plots intersected practically on the abscissa following EGFR-TK pre-activation with EGF, but below the abscissa without EGF pre-activation. The EGFR-TK inhibitors App(NH)p (5'-adenylyl-beta, gamma-imidodiphosphate) and ADP were competitive with ATP and noncompetitive with GAT. Four families of 1/v vs. 1/[ATP] plots, constructed at different fixed concentrations of ADP and a different constant concentration of GAT for each family, yielded Slope1/ATP replots which intersected to the left of the ordinate and below the abscissa. GAT and AT, as cosubstrates, were competitive with each other and noncompetitive with ATP; 1/v vs. 1/[GAT] or 1/[AT] plots were hyperbolic and reached horizontal asymptotes when v was expressed as the rate of common product formation. All data were subjected to computer best-fit analysis by a program written especially for this purpose. We conclude that (i) the EGFR-TK reaction follows a Sequential Bi-Bi Rapid Equilibrium Random mechanism, and (ii) EGF induces conformational changes in the EGFR-TK active center which lead to marked decreases in the apparent dissociation constants of both substrates of the kinase reaction and a concomitant increase in initial velocities and Vmax (apparent).     

The imbalance of brain large-chain aminoacid availability in amyotrophic lateral sclerosis patients treated with high doses of branched-chain aminoacids

Following the report of an increased mortality among patients with amyotrophic lateral sclerosis given high daily doses of branched-chain aminoacids, we assessed the plasma concentrations of large neutral aminoacids and glutamic acid and the large neutral aminoacid brain influx in 24 amyotrophic lateral sclerosis patients receiving placebo or branched-chain aminoacids ( -leucine 12 g, -isoleucine 6 g, -valine 6 g daily), in 15 untreated amyotrophic lateral sclerosis patients and in 15 healthy volunteers. The branched-chain aminoacid plasma concentrations increased three- to six-fold in the treated group compared to the patients receiving placebo or no treatment and to the healthy controls. Plasma glutamic acid concentrations in healthy volunteers were 51.59±7.53 nmol/ml while in the amyotrophic lateral sclerosis patients receiving no treatment, placebo or branched-chain aminoacids were 92.33±12.15 nmol/ml, 91.21±15.86 nmol/ml and 95.08±17.96 nmol/ml respectively. The glutamic acid concentration was significantly higher (P<0.01) in amyotrophic lateral sclerosis patients than in healthy individuals. Plasma phenylalanine and tyrosine were lower in the amyotrophic lateral sclerosis patients than in healthy controls, regardless of treatment, whereas tryptophan levels were not significantly different. The branched-chain aminoacid brain influx of the treated group was 110-140% of that measured in the patients receiving placebo and in the healthy controls. The aromatic aminoacid brain influx was lower in the treated group than in the placebo group or healthy controls. An impairment of brain large neutral aminoacid availability might possible contribute to enhancing the progression of symptoms in patients with amyotrophic lateral sclerosis.     

Steady-state kinetic mechanism of Ras farnesyl:protein transferase.

The steady-state kinetic mechanism of bovine brain farnesyl:protein transferase (FPTase) has been determined using a series of initial velocity studies, including both dead-end substrate and product inhibitor experiments. Reciprocal plots of the initial velocity data intersected on the 1/[s] axis, indicating that a ternary complex forms (sequential mechanism) and suggesting that the binding of one substrate does not affect the binding of the other. The order of substrate addition was probed by determining the patterns of dead-end substrate and product inhibition. Two nonhydrolyzable analogues of farnesyl diphosphate, (alpha-hydroxyfarnesyl)phosphonic acid (1) and [[(farnesylmethyl)hydroxyphosphinyl]methyl]phosphonic acid (2), were both shown to be competitive inhibitors of farnesyl diphosphate and noncompetitive inhibitors of Ras-CVLS. Four nonsubstrate tetrapeptides, CV[D-L]S, CVLS-NH2, N-acetyl-L-penicillamine-VIM, and CIFM, were all shown to be noncompetitive inhibitors of farnesyl diphosphate and competitive inhibitors of Ras-CVLS. These data are consistent with random order of substrate addition. Product inhibition patterns corroborated the results found with the dead-end substrate inhibitors. We conclude that bovine brain FPTase proceeds through a random order sequential mechanism. Determination of steady-state parameters for several physiological Ras-CaaX variants showed that amino acid changes affected the values of KM, but not those of kcat, suggesting that the catalytic efficiencies (kcat/KM) of Ras-CaaX substrates depend largely upon their relative binding affinity for FPTase.     

On the donor substrate dependence of group-transfer reactions by hydrolytic enzymes: Insight from kinetic analysis of sucrose phosphorylase-catalyzed transglycosylation

Chemical group-transfer reactions by hydrolytic enzymes have considerable importance in biocatalytic synthesis and are exploited broadly in commercial-scale chemical production. Mechanistically, these reactions have in common the involvement of a covalent enzyme intermediate which is formed upon enzyme reaction with the donor substrate and is subsequently intercepted by a suitable acceptor. Here, we studied the glycosylation of glycerol from sucrose by sucrose phosphorylase (SucP) to clarify a peculiar, yet generally important characteristic of this reaction: partitioning between glycosylation of glycerol and hydrolysis depends on the type and the concentration of the donor substrate used (here: sucrose, α-d -glucose 1-phosphate (G1P)). We develop a kinetic framework to analyze the effect and provide evidence that, when G1P is used as donor substrate, hydrolysis occurs not only from the β-glucosyl-enzyme intermediate (E-Glc), but additionally from a noncovalent complex of E-Glc and substrate which unlike E-Glc is unreactive to glycerol. Depending on the relative rates of hydrolysis of free and substrate-bound E-Glc, inhibition (Leuconostoc mesenteroides SucP) or apparent activation (Bifidobacterium adolescentis SucP) is observed at high donor substrate concentration. At a G1P concentration that excludes the substrate-bound E-Glc, the transfer/hydrolysis ratio changes to a value consistent with reaction exclusively through E-Glc, independent of the donor substrate used. Collectively, these results give explanation for a kinetic behavior of SucP not previously accounted for, provide essential basis for design and optimization of the synthetic reaction, and establish a theoretical framework for the analysis of kinetically analogous group-transfer reactions by hydrolytic enzymes.     

Kinetic mechanism and identification of the active site tyrosine residue in Enterobacter amnigenus arylsulfate sulfotransferase.

Bacterial arylsulfate sulfotransferase (ASST) catalyzes the transfer of a sulfate group from a phenyl sulfate ester to a phenolic acceptor. The kinetic mechanism of Enterobacter amnigenus ASST was determined. Plots of 1/v versus 1/[substrate (A)] at different fixed substrate (B) concentrations gave a series of parallel lines. One of the reaction products, p-nitrophenol, inhibited the enzyme noncompetitively with respect to p-nitrophenyl sulfate, but competitively to alpha-naphthol. These results correspond to a ping pong bi bi mechanism. By site-directed mutagenesis, we substituted each conserved tyrosine residue with phenylalanine. Among the mutants, Y123F showed severely reduced catalytic activity. We conclude that Tyr 123 is an essential active site residue. A mechanistic hypothesis is presented to account for these observations.     

Cerebral-cortex hexokinases. Elucidation of reaction mechanisms by substrate and dead-end inhibitor kinetic analysis

1. The substrate kinetic properties of cerebral hexokinases (mitochondrial and cytoplasmic) were studied at limiting concentrations of both glucose and MgATP(2-). Primary plots of the enzymic activity gave no evidence of a Ping Pong mechanism in three types of mitochondrial preparation tested (intact and osmotically disrupted mitochondria, and the purified mitochondrial enzyme), nor in the purified cytoplasmic preparation. 2. Secondary plots of intercepts from the primary plots (1/v versus 1/s) versus reciprocal of second substrate of the mitochondrial activity gave kinetic constants which differed from those obtained directly from the plots of 1/v versus 1/s or of s/v versus s, although the ratios of the derived constants were consistent. The kinetic constants obtained with the cytoplasmic enzyme from primary and secondary plots were consistent. 3. Deoxyglucose, as alternative substrate, inhibited cytoplasmic hexokinase by competition with glucose, but did not compete when MgATP(2-) was the substrate varied. The K(i) for deoxyglucose when glucose concentrations were varied was 0.25mm. 4. A range of ATP analogues was tested as potential substrates and inhibitors of hexokinase activity. GTP, ITP, CTP, UTP and betagamma-methylene-ATP did not act as substrates, nor did they cause significant inhibition. Deoxy-ATP proved to be almost as effective a substrate as ATP. AMP inhibited but did not act as substrate. 5. N-Acetyl-glucosamine inhibited all preparations competitively when glucose was varied and non-competitively when MgATP(2-) was varied. AMP inhibition was competitive when MgATP(2-) was the substrate varied and non-competitive when glucose was varied. 6. The results are interpreted as providing evidence for a random reaction mechanism in all preparations of brain hexokinase, cytoplasmic and mitochondrial. The kinetic properties and reaction mechanism do not change on extraction and purification of the particulate enzyme. 7. The results are discussed in terms of the participation of hexokinase in regulation of cerebral glycolysis.     

Kinetics of enzymes subject to very strong product inhibition: Analysis using simplified integrated rate equations and average velocities

Enzymes which catalyze energetically unfavorable reactions in the physiological direction are likely to be strongly inhibited by the reaction products. (Some energetically favorable reactions may also display strong "product inhibition" when assayed in the reverse direction.) In some cases, the inhibition caused by an accumulating product is so potent that true initial velocities cannot be directly determined using conventional assay methods. Continuous removal of the inhibitory product may be mitigated against by the nature of the assay or the unavailability of the appropriate coupling enzyme. It can be shown that if (a) only one inhibitory product is allowed to accumulate and (b) the substrate concentrations remain essentially constant over the assay period (i.e. Kproduct less than or equal to 10(-2)Ksubstrate, so that the decreasing reaction rate stems only from progressive product inhibition), then plots of reciprocal average (apparent) velocity (i.e. 1/v = t/[P]) versus [P] are linear and extrapolate to 1/v0, the reciprocal of the initial uninhibited velocity at the fixed substrate concentrations. Intercept replots give the usual initial velocity reciprocal plot patterns and permit Vmax and the substrate Km's to be determined. Slope replots are diagnostic of the type of inhibition exerted by the accumulating product and permit the inhibition constants to be determined. If all the appropriate coupling enzymes are available, some kinetic mechanisms can be diagnosed using data derived from the reaction progress curves in the presence of one accumulating product at a time.     

Reduction of 2,4,6-trinitrobenzenesulfonate by glutathione reductase and the effect of NADP+ on the electron transfer

Glutathione reductase has been found to catalyze an NAD(P)H-dependent electron transfer to 2,4,6-trinitrobenzenesulfonate (TNBS). In the presence of oxygen TNBS is not consumed in the reaction, but is rapidly reoxidized with concomitant production of hydrogen peroxide. Cytochrome c can replace oxygen as the final electron acceptor, indicating that a one-electron transfer takes place. The rate is slightly higher in the absence than in the presence of oxygen, ruling out superoxide anion as an obligatory intermediate in cytochrome c reduction. In the absence of oxygen (or cytochrome c), TNBS limits the reaction and accepts a total of four electrons. The TNBS-dependent NADPH (or NADH) oxidation is markedly stimulated by NADP+, and to a smaller extent also by NAD+. The TNBS-dependent reactions are inhibited by excess of NADPH but not by NADH. The kinetics of these reactions are consistent with a branching reaction mechanism in which a pathway including a ternary complex between the two-electron reduced enzyme and NADP+ has the highest turnover. NADPH-dependent reductions of ferricyanide or 2,6-dichloroindophenol catalyzed by glutathione reductase are also markedly influenced by NADP+. Evidently NADP+ facilitates a shift of the catalyzed reaction from the normal two-electron reduction of glutathione disulfide to a more unspecific one-electron reduction of other acceptors. Spectral as well as kinetic data suggest that the rate of radical formation limits the reactions with the artificial electron acceptors and that NADP+ promotes this rate-limiting step.     

Intermembrane transport and antioxidant action of alpha-tocopherol in liposomes

Studies were made of the ability of alpha-tocopherol, incorporated into unilamellar liposomes from saturated or unsaturated phospholipids (donor liposomes) to inhibit the accumulation of lipid peroxidation (LPO) products in unilamellar liposomes from rat cerebral cortex lipids (acceptor liposomes) in the presence of LPO inducer (Fe + ascorbate). With the molar alpha-tocopherol: phospholipids rations from 1:1000 to 1:100 in donor liposomes, obtained through sonication of lipid dispersions, alpha-tocopherol was incorporated into both monolayers of liposomes and was distributed in monomeric form without forming clusters. Based on the dependencies of LPO inhibition on the alpha-tocopherol concentrations, we chose the ones that completely prevented the accumulation of LPO products in donor liposomes. Under these conditions LPO inhibition in mixtures of donor and acceptors liposomes was fully determined by the antioxidant effect of alpha-tocopherol in acceptor liposomes due to its intermembrane transfer. The efficiency of the "intermembrane" antioxidant action of alpha-tocopherol increased in the course of preincubation of donor and acceptor liposomes (up to 60 min) and this increase was more pronounced when the donor liposomes contained unsaturated phospholipids. Evidence was obtained that the intermembrane transfer of alpha-tocopherol did not result from the fusion of donor and acceptor liposomes during preincubation.     

Primary photochemistry in the facultative green photosynthetic bacterium Chloroflexus aurantiacus

The mechanism of primary photochemistry has been investigated in purified cytoplasmic membranes and isolated reaction centers of Chloroflexus aurantiacus. Redox titrations on the cytoplasmic membranes indicate that the midpoint redox potential of P870, the primary electron donor bacteriochlorophyll, is +362 mV. An early electron acceptor, presumably menaquinone has Em 8.1 = -50 mV, and a tightly bound photooxidizable cytochrome c554 has Em 8.1 = +245 mV. The isolated reaction center has a bacteriochlorophyll to bacteriopheophytin ratio of 0.94:1. A two-quinone acceptor system is present, and is inhibited by o-phenanthroline. Picosecond transient absorption and kinetic measurements indicate the bacteriopheophytin and bacteriochlorophyll form an earlier electron acceptor complex.     

Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: dependence of the rate on the composition of the redox buffer

The velocity of the oxidative renaturation of reduced ribonuclease A catalyzed by protein disulfide isomerase (PDI) is strongly dependent on the composition of a glutathione/glutathione disulfide redox buffer. As with the uncatalyzed, glutathione-mediated oxidative folding of ribonuclease, the steady-state velocity of the PDI-catalyzed reaction displays a distinct optimum with respect to both the glutathione (GSH) and glutathione disulfide (GSSG) concentrations. Optimum activity is observed at [GSH] = 1.0 mM and [GSSG] = 0.2 mM. The apparent kcat at saturating RNase concentration is 0.46 +/- 0.05 mumol of RNase renatured min-1 (mumol of PDI)-1 compared to the apparent first-order rate constant for the uncatalyzed reaction of 0.02 +/- 0.01 min-1. Changes in GSH and GSSG concentration have a similar effect on the rate of both the PDI-catalyzed and uncatalyzed reactions except under the more oxidizing conditions employed, where the catalytic effectiveness of PDI is diminished. The ratio of the velocity of the catalyzed reaction to that of the uncatalyzed reaction increases as the quantity [GSH]2/[GSSG] increases and approaches a constant, limiting value at [GSH]2/[GSSG] greater than 1 mM, suggesting that a reduced, dithiol form of PDI is required for optimum activity. As long as the glutathione redox buffer is sufficiently reducing to maintain PDI in an active form [( GSH]2/[GSSG] greater than 1 mM), the rate acceleration provided by PDI is reasonably constant, although the actual rate may vary by more than an order of magnitude. PDI exhibits half of the maximum rate acceleration at a [GSH]2/[GSSG] of 0.06 +/- 0.01 mM.