Rate-limiting Transfer Research Articles

Diazinon Interrupts Ovarian Steroidogenic Acute Regulatory (StAR) Gene Transcription in Gonadotropin-Stimulated Rat Model.

Organophosphate pesticides are considered as endocrine disruptors that interfere with reproductive functions. The corpus luteum (CL) is a transient endocrine gland that produces progesterone, a crucial hormone for a successful beginning and maintenance of pregnancy. Steroidogenic acute regulatory protein (StAR) facilitates the rate-limiting transfer of cholesterol from the outer mitochondrial membrane to the inner organelle membranes. We investigated the effect of Diazinon (DZN), an organophosphate, on StAR mRNA expression by Sybergreen Real Time-PCR in a time-dependent manner in luteal phase. Fifty immature female Wistar rats (24-day-old) were injected with a single injection of Pregnant mare's Serum Gonadotropin (PMSG) followed by a single injection of human Chorionic Gonadotropin (hCG), 48 h later. Then, DZN was administered in a single dose (70 mg/kg bw, I.P), controls received only the vehicle, 12 h post-hCG injection. Ovaries were collected in 4 h. intervals from 8 to 24 h post-hCG injection. Then, hCG stimulation transcript levels of StAR gene were significantly altered in the hormone-stimulated rats following DZN treatment. In addition, histological study showed that the CL diameter in DZN-treated group was smaller than control group (p = 0.000). Our findings suggest that the critical step in the function of CL is disrupted by DZN and may correlates with female reproductive damage.

Enhanced Rate Performance in Electrode Materials for Lithium-Ion Batteries

Lithium-ion batteries are dominating the field of energy storage and have already started to enter the electric vehicle market. This market is striving for better electrochemical performance, such as higher specific energy and energy density, longer lifetime and improved safety. In order to fulfil these challenges, the exploration of new materials is usually preferred. Through this study we prove that electrode design using established anode chemistry is a powerful tool to enhance the performance of Li ion batteries. One of the main limitations of current batteries lies in the transport of lithium ions, especially at high cycling rates and in high loading electrodes. This shortcoming does not arise from the insertion/extraction kinetics within the bulk active particles [1] but from the slow diffusion of lithium ions across the electrode. In particular, reduction of the tortuosity would accelerate such rate-limiting transfer process [2]. Minimising the ion and electron path tortuosity within thick electrodes would lead to batteries capable of sustaining higher cycling rates and power densities. We used a combination of electrochemical measurements, 3D imaging and diffusion modelling by Finite Element Analysis to study the charge carrier transport properties in architectured electrodes. Through this presentation we will show that advanced electrode engineering can be used to greatly improve the electrochemical performance of anodes for lithium-ion batteries. Tortuosity of electrode materials is significantly decreased in favor of higher specific charges (up to three times) at high cycling rates.

Molecular basis of intramolecular electron transfer in proteins during radical-mediated oxidations: Computer simulation studies in model tyrosine–cysteine peptides in solution

Experimental studies in hemeproteins and model Tyr/Cys-containing peptides exposed to oxidizing and nitrating species suggest that intramolecular electron transfer (IET) between tyrosyl radicals (Tyr-O) and Cys residues controls oxidative modification yields. The molecular basis of this IET process is not sufficiently understood with structural atomic detail. Herein, we analyzed using molecular dynamics and quantum mechanics-based computational calculations, mechanistic possibilities for the radical transfer reaction in Tyr/Cys-containing peptides in solution and correlated them with existing experimental data. Our results support that Tyr-O to Cys radical transfer is mediated by an acid/base equilibrium that involves deprotonation of Cys to form the thiolate, followed by a likely rate-limiting transfer process to yield cysteinyl radical and a Tyr phenolate; proton uptake by Tyr completes the reaction. Both, the pKa values of the Tyr phenol and Cys thiol groups and the energetic and kinetics of the reversible IET are revealed as key physico-chemical factors. The proposed mechanism constitutes a case of sequential, acid/base equilibrium-dependent and solvent-mediated, proton-coupled electron transfer and explains the dependency of oxidative yields in Tyr/Cys peptides as a function of the number of alanine spacers. These findings contribute to explain oxidative modifications in proteins that contain sequence and/or spatially close Tyr–Cys residues.

Transcription of Steroidogenic Acute Regulatory Protein in the Rodent Ovary and Placenta: Alternative Modes of Cyclic Adenosine 3′, 5′-Monophosphate Dependent and Independent Regulation

Steroid hormone synthesis is a vital function of the adrenal cortex, serves a critical role in gonadal function, and maintains pregnancy if normally executed in the placenta. The substrate for the synthesis of all steroid hormones is cholesterol, and its conversion to the first steroid, pregnenolone, by the cholesterol side-chain cleavage cytochrome P450 (CYP11A1) enzyme complex takes place in the inner mitochondrial membranes. Steroidogenic acute regulatory protein (STAR) facilitates the rate-limiting transfer of cholesterol from the outer mitochondrial membrane to CYP11A1 located in the inner organelle membranes. The current study explored the mechanisms controlling transcription of the Star gene in primary cell cultures of mouse placental trophoblast giant cells and rat ovarian granulosa cells examined throughout the course of their functional differentiation. Our findings show that the cis-elements required for Star transcription in the rodent placenta and the ovary are centered in a relatively small proximal region of the promoter. In placental trophoblast giant cells, cAMP is required for activation of the Star promoter, and the cis-elements mediating a maximal response were defined as cAMP response element 2 and GATA. EMSA studies show that placental cAMP-responsive element binding protein (CREB)-1 and activating transcription factor-2 (ATF2) bind to a -81/-78 sequence, whereas GATA-2 binds to a -66/-61 sequence. In comparison, patterns of Star regulation in the ovary suggested tissue-specific and developmental controlled modes of Star transcription. During the follicular phase, FSH/cAMP induced CREB-1 dependent activity, whereas upon luteinization STAR expression becomes cAMP and CREB independent, a functional shift conferred by FOS-related antigen-2 displacement of CREB-1 binding, and the appearance of a new requirement for CCAAT enhancer-binding protein beta and steroidogenic factor 1 that bind to upstream elements (-117/-95). These findings suggest that during evolution, the promoters of the Star gene acquired nonconsensus sequence elements enabling expression of a single gene in different organs, or allowing dynamic temporal changes corresponding to progressing phases of differentiation in a given cell type.

Chlorine Dioxide Oxidation of Dihydronicotinamide Adenine Dinucleotide (NADH)

The oxidation of dihydronicotinamide adenine dinucleotide (NADH) by chlorine dioxide in phosphate buffered solutions (pH 6-8) is very rapid with a second-order rate constant of 3.9 x 10(6) M(-1) s(-1) at 24.6 degrees C. The overall reaction stoichiometry is 2ClO2(*) per NADH. In contrast to many oxidants where NADH reacts by hydride transfer, the proposed mechanism is a rate-limiting transfer of an electron from NADH to ClO2(*). Subsequent sequential fast reactions with H(+) transfer to H2O and transfer of an electron to a second ClO2(*) give 2ClO2(-), H3O(+), and NAD(+) as products. The electrode potential of 0.936 V for the ClO2(*)/ClO2(-) couple is so large that even 0.1 M of added ClO2(-) (a 10(3) excess over the initial ClO2(*) concentration) fails to suppress the reaction rate.

How Do Enzymes Activate Oxygen Without Inactivating Themselves?

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How Do Enzymes Activate Oxygen without Inactivating Themselves?

Detailed analyses of the oxidative half-reactions of glucose oxidase and soybean lipoxygenase provide insight into Nature's solution to the "trouble with oxygen". Coupled with studies of other O2-activating enzymes, two key features emerge. The first is the predominance of a rate-limiting transfer of the first electron transfer to O2, with subsequent electron and proton transfers occurring in rapid steps. The second feature is the identification of non-metal binding sites and channels for O2. These permit a controlled reactivity of oxygen to generate the desired regio- and stereochemical products, while minimizing deleterious side reactions.

Carbenoid Chain Reactions: Substitutions by Organolithium Compounds at Unactivated 1-Chloro-1-alkenes

The deceptively simple "cross-coupling" reactions Alk(2)C=CA-Cl + RLi --> Alk(2)C=CA-R + LiCl (A = H, D, or Cl) occur via an alkylidenecarbenoid chain mechanism in three steps without a transition metal catalyst. In the initiating step 1, the sterically shielded 2-(chloromethylidene)-1,1,3,3-tetramethylindans 2a-c (Alk(2)C=CA-Cl) generate a Cl,Li-alkylidenecarbenoid (Alk(2)C=CLi-Cl, 6) through the transfer of atom A to RLi (methyllithium, n-butyllithium, or aryllithium). The chain cycle consists of the following two steps: (i) A fast vinylic substitution reaction of these RLi at carbenoid 6 (step 2) with formation of the chain carrier Alk(2)C=CLi-R (8), and (ii) a rate-limiting transfer of atom A (step 3) from reagent 2 to the chain carrier 8 with formation of the product Alk(2)C=CA-R (4) and with regeneration of carbenoid 6. This chain propagation step 3 was sufficiently slow to allow steady-state concentrations of Alk(2)C=CLi-Aryl to be observed (by NMR) with RLi = C6H5Li (in Et2O) and with 4-(Me3Si)C6H4Li (in t-BuOMe), whereas these chain processes were much faster in THF solution. PhC[triple bond]CLi cannot perform step 1, but its carbenoid chain processes with reagents 2a and 2c may be started with MeLi, whereafter LiC[triple bond]CPh reacts faster than MeLi in the product-determining step 2 to generate the chain carrier Alk(2)C=CLi-C[triple bond]CPh (8g), which completes its chain cycle through the slower step 3. The sterically congested products were formed with surprising ease even with RLi as bulky as 2,6-dimethylphenyllithium and 2,4,6-tri-tert-butylphenyllithium.

Guanidine Derivatives Rescue the Arg418Ala Mutation of Tritrichomonas foetus IMP Dehydrogenase

IMP dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) and the reduction of NAD(+). The reaction involves formation of an E-XMP covalent intermediate; hydrolysis of the E-XMP intermediate is rate-limiting and requires the enzyme to adopt a closed conformation. Arg418 appears to act as the base that activates water for the hydrolysis reaction [Guillen-Schlippe, Y. V., and Hedstrom, L. (2005) Biochemistry 44, 11700-11707]. Deprotonation of Arg418 also stabilizes the closed conformation. Here we show that guanidine derivatives rescue the activity of the Arg418Ala variant. Amines and imidazole do not rescue. The rescue reaction appears to be saturable, with the values of K(R) ranging from 40 to 400 mM. The value of k(rescue) for the best rescue agents approaches the value of k(cat) for the reaction of the wild-type enzyme. Guanidine derivatives also rescue the activity of the Arg418Ala/Tyr419Phe variant. Multiple-inhibitor experiments suggest that the guanidine derivatives do not restore the equilibrium between open and closed conformations. Therefore, rescue agents must accelerate the hydrolysis of the E-XMP intermediate. The rate of the rescue reaction increases with an increase in pH, consistent with the hypothesis that the reaction involves neutral guanidine. A solvent D(2)O isotope effect is observed at low concentrations of the rescue agent, consistent with rate-limiting transfer of a proton from water. The value of k(cat) (rescue)/K(R)(base) correlates with the pK(a) of the guanidine derivative (Bronsted coefficient beta approximately 1). These results suggest that proton transfer from water to guanidine is almost complete in the transition state.

Transfer of Cholesterol between Phospholipid Vesicles Mediated by the Steroidogenic Acute Regulatory Protein (StAR)

The steroidogenic acute regulatory protein (StAR) mediates the acute stimulation of steroid synthesis by tropic hormones in steroidogenic cells. StAR interacts with the outer mitochondrial membrane and facilitates the rate-limiting transfer of cholesterol to the inner mitochondrial membrane where cytochrome P-450scc converts this cholesterol into pregnenolone. We tested the ability of N-62 StAR to transfer cholesterol from donor vesicles containing cholesterol but no cytochrome P-450scc to acceptor vesicles containing P-450scc but no cholesterol, using P-450scc activity as a reporter of the cholesterol content of synthetic phospholipid vesicles. N-62 StAR stimulated P-450scc activity in acceptor vesicles 5-10-fold following the addition of donor vesicles. Transfer of cholesterol to acceptor vesicles was rapid and sufficient to maintain a linear rate of pregnenolone synthesis for 10 min. The effect of N-62 StAR in stimulating P-450scc activity was specific for cholesterol transfer and was not due to vesicle fusion or P-450scc exchange between vesicles. Maximum stimulation of P-450scc activity in acceptor vesicles required preincubation of N-62 StAR with phospholipid vesicles prior to adding donor vesicles. The amount of N-62 StAR causing half-maximum stimulation of P-450scc activity in acceptor vesicles was 1.9 microm. Half-maximum stimulation required more than a 10-fold higher concentration of R182L N-62 StAR, a mutant associated with congenital lipoid adrenal hyperplasia. N-62 StAR-mediated transfer of cholesterol between vesicles showed low dependence on the cholesterol concentration in the donor vesicles. Thus StAR can transfer cholesterol between synthetic membranes without other protein components found in mitochondria.

Catalysis of the oxidation of NADH by heteropolyanions: a kinetic study

Abstract The oxidation of NADH is readily obtained in the presence of Dawson-type heteropolyanions (HPA). A stoichiometry of 2:1 is established for the oxometalate/NADH couple. The starring system for electrochemistry studies consists of the one-electron reduction product of the heteropolyanions in the presence of various amounts of NADH. The quantitative study by cyclic voltammetry confirms that the oxidized form of the selected HPAs are capable of catalyzing the oxidation of NADH. The kinetics were studied quantitatively by double-step chronocoulometry. The logarithm of the second-order rate constant varies linearly with the E o of the first redox system of the HPAs with a slope of 16.4 V −1 , indicating that the oxidation of NADH proceeds by a multistep mechanism involving an initial rate-limiting one-electron transfer. The E o value for the NADH/ NADH ·+ redox couple has been estimated.

Studies of 8-Azido-ATP Adducts Reveal Two Mechanisms by Which ATP Binding to Cytochrome c Could Inhibit Respiration

We have proposed that the binding of ATP at a site of substantial affinity and specificity could regulate the activity of cytochrome c with its physiological partners and thus the overall efficiency of mitochondrial electron transport. We now describe the use of ATP affinity-labeled protein to test the effect of occupancy of that site, which includes the invariant arginine 91, on the activity of cytochrome c with purified cytochrome c reductase and oxidase and its association with the mitochondrial inner membrane. Electron-transfer activities with the reductase and oxidase were inhibited by site occupancy to 41% and 11-15% of native values, respectively. The marked difference in the degree of inhibition of activity that distinguishes the reactions with the two major physiological partners was sufficient to cause, in whole mitochondria, a demonstrable shift from a situation in which there is a rate-limiting transfer from the reductase to cytochrome c, to a state where rates are more evenly matched for transfers between cytochrome c and the two redox partners. Site occupancy also substantially reduces the ionic strength necessary for half-maximal dissociation of cytochrome c from the membrane. These data imply that the decreased efficiency of electron transfer caused by ATP attachment can be attributed to a decrease in the protein's activity with individual physiological partners, possibly compounded with a decrease in its affinity for the inner mitochondrial membrane, and suggest that feedback regulation by ATP of cellular respiration operates in like manner.

Human Placental Biotin Transport: Normal Characteristics and Effect of Ethanol

Biotin, a vitamin essential for many metabolic reactions, is supplied to the fetus exclusively from the mother. Deficiency of biotin in pregnancy leads to impaired fetal growth and development. Alcohol taken in pregnancy likewise may cause fetal growth abnormalities. Normal biotin transport via the placenta and the effects of ethanol on this transport apparently have not been studied. Our aims were to characterize these phenomena for the normal human-term placenta. Using maternal-facing placental membrane vesicles, biotin uptake was sodium- and temperature-dependent, saturable, and inhibited by structural analogs of biotin (desthiobiotin, biocytin, and biotin methyl ester), as well as by 4 and 10 hr exposure to 3 g/liter ethanol. Using the isolated perfused single cotyledon method to measure placental transport of biotin at a perfusion concentration of 1 nM, the overall rate of biotin transport was found to be only 30% that of antipyrine, a freely diffusible marker. Clearance of biotin was approximately 2 ml/hr.g placenta, which was equal to the clearance of passively transferred L-glucose; biotin clearance was similar in both maternal to fetal and fetal to maternal directions. Overall transfer of biotin from maternal to fetal compartments was not inhibited by 500-fold greater concentrations of the three analogs, did not proceed against a biotin concentration gradient, and was not inhibited by 90-240 min exposure to an initial concentration of 4 g/liter ethanol. Concentration of biotin in the fetal compartment at the end of the study was not higher than on the maternal side (after maternal to fetal infusion), but placental concentration was 2- to 3-fold greater. No significant metabolism of biotin was detected. Exposing human placental cultured trophoblast on day 3 to 24 hr of ethanol (2 g/liter) had no effect on the net uptake of biotin by these cells. These studies provide evidence that maternal-facing placental membranes take up biotin by a mediated, carrier-dependent process that is inhibited by ethanol; however, based on the perfusion studies, we conclude that the overall (maternal-fetal) rate-limiting transfer of biotin by the human placenta is most consistent with a passive process, which is not inhibited by short-term exposure to ethanol.

Protein synthesis, cell growth and oncogenesis

Two lines of investigation support a new hypothesis concerning the role of protein synthesis in the mitogenic pathway. The first is that a variety of mitogens and oncogene products increase phosphorylation and thereby activate eIF-4E, which is involved in the rate-limiting transfer of mRNA to ribosomes. The second is that overexpression or microinjection of eIF-4E induce rapid cell proliferation and oncogenic transformation.

Transport of neutral species in electroactive polymer films

A new procedure has been applied to the analysis of electrochemical quartz crystal microbalance data for transfer of mobile species in electroactive polymer films. The procedure enables the distinction between global equilibrium and rate-limiting transfer of electrons, ions and neutral species during film redox conversion. For polyvinylferrocene, polythionine and polybithiophene films (under the experimental conditions employed), transfer of a neutral species was found to be rate limiting. In the first two cases, solvent (water) was unequivocally identified as the slow species. In the third case, solvent (CH3CN) transfer was tentatively identified as the rate-limiting step. Slow transfer of neutral species is due to the absence of migration effects, which can aid ion transfer. Transfer rates depend on direction of transfer in all three systems.

Effect of glycerol on gluconeogenesis in isolated rabbit kidney cortex tubules

In renal tubules isolated from fed rabbits glycerol is not utilized as a glucose precursor, probably due to the rate-limiting transfer of reducing equivalents from cytosol to mitochondria. Pyruvate and glutamate stimulated an incorporation of [ 14C]glycerol to glucose by 50- and 10-fold, respectively, indicating that glycerol is utilized as a gluconeogenic substrate under these conditions. Glycerol at concentration of 1.5 mM resulted in an acceleration of both glucose formation and incorporation of [ 14C]pyruvate and [ 14C]glutamate into glucose by 2- and 9-fold, respectively, while it decreased the rates of these processes from lactate as a substrate. In the presence of fructose, glycerol decreased the ATP level, limiting the rate of fructose phosphorylation and glucose synthesis. As concluded from the ‘cross-over’ plots, the ratios of both 3-hydroxybutyrate/acetoacetate and glycerol 3-phosphate/dihydroxyacetone phosphate, as well as from experiments performed with methylene blue and acetoacetate, the stimulatory effect of glycerol on glucose formation from pyruvate and glutamate may result from an acceleration of fluxes through the first steps of gluconeogenesis as well as glyceraldehyde-3-phosphate dehydrogenase. As inhibition by glycerol of gluconeogenesis from lactate is probably due to a marked elevation of the cytosolic NADH/NAD + ratio resulting in a decline of flux through lactate dehydrogenase.

The biosynthesis of rat transferrin. Evidence for rapid glycosylation, disulfide bond formation, and tertiary folding.

The transit time of newly synthesized transferrin in the liver is markedly longer than that of albumin. We sought to learn the basis of this difference by the use of labeled leucine and mannose in vivo and by isolation of newly formed transferrin from rough microsomes of rat liver. Albumin and alpha 1-antitrypsin, a second glycoprotein, were also studied for comparison. Minimal hepatic transit times were 17, 23, and 31 min for albumin, alpha 1-antitrypsin, and transferrin, respectively. The delay in the case of transferrin was found to occur chiefly in the rough endoplasmic reticulum and to be paralleled by an increase in the amount of transferrin relative to albumin in that organelle. Initial glycosylation of transferrin was as rapid as that of alpha 1-antitrypsin, and essentially all of the transferrin in the rough endoplasmic reticulum contained glycans which bound to concanavalin A and were removed by endoglycosidase H. Only 3% of the transferrin isolated from the rough microsomes came from the plasma by endocytosis or adsorption. Rapidity of disulfide bond formation in rough microsomes was evident from the presence of only 1.3 cysteine thiols/molecule of rough microsomal transferrin (total of 19 cystines) and the absence of mixed disulfides. Peptide patterns upon mild proteolysis were consistent with a native configuration of disulfide bond pairing. The ability of rough microsomal transferrin to bind and deliver iron through interaction with transferrin receptors on reticulocytes suggests that considerable tertiary structure is present. Thus, initial glycosylation, disulfide bridging, and tertiary folding are all rapid processes. The cause for the slow release of transferrin from the rough endoplasmic reticulum may lie with a rate-limiting transfer mechanism.

Picosecond study of energy-transfer kinetics in phycobilisomes of Synechococcus 6301 and the mutant AN 112

Excitation-energy-transfer kinetics in isolated phycobilisomes from the cyanobacterium Synechococcus 6301 ( Anacystis nidulans) and the mutant AN 112 (rods containing one hexameric C-phycocyanin unit only) was investigated by picosecond absorption and fluorescence techniques. The different chromophores in the phycobilisomes were selectively excited. A lifetime component of about 10 ps was found for both C-phycocyanin and allophycocyanin in both types of phycobilisomes. We assign these signals to a transfer of excitation energy from sensitizing (‘s’) to fluorescing (‘f’) chromophores within C-phycocyanin and allophycocyanin units. A 10 ps component was also observed in the anisotropy relaxation measurements. The anisotropy decay is attributed mainly to differently oriented transition dipole moments of ‘s’- and ‘f’-chromophores and partially to ‘f’ → ‘f’ transfer. An absorption recovery signal of τ ≈ 90 ps at λ ≤ 630 nm in phycobilisomes of Synechococcus 6301 is reduced to 40–50 ps in AN 112 phycobilisomes. This is rationalized in terms of a decreased rod → core transfer time in the shorter rods of AN 112. The 40–50 ps lifetime of fluorescence and absorption recovery in AN 112 phycobilisomes is assigned mainly to a rate-limiting transfer step between C-phycocyanin and the allophycocyanin core. A decay component of allophycocyanin τ ≈ 50 ps was observed both in absorption recovery measurements and in fluorescence decay. It is assigned to energy transfer to the terminal chromophores. The final emitter(s) of the phycobilisomes from AN 112 have fluorescence lifetimes of 1.9 and 1.3 ns. We find a good correlation in the fluorescence kinetics between the decay times of phycocyanin and allophycocyanin and the fluorescence risetimes of the terminal emitters.

Simultaneous proton transfers coupled with heavy atom motion in the transition state of the 2-pyridone-catalysed mutarotation of tetramethylglucose. Primary and secondry deuterium isotope effects

Primary and secondary deuterium isotope effects and calculated primary isotope effects for the 2-pyridone-catalysed mutarotation of tetramethylglucose suggest rate-limiting simultaneous transfer of two protons coupled with considerable heavy atom motion.

Transition-state structure in the yeast alcohol dehydrogenase reaction: the magnitude of solvent and alpha-secondary hydrogen isotope effects.

Solvent and alpha-secondary isotope effects have been measured in the yeast alcohol dehydrogenase reaction, under conditions of a rate-limiting transfer of hydrogen between coenzyme and substrate. Determination of catalytic constants (at saturating concentrations of substrate and coenzyme) in H2O and D2O as a function of pH(D) has allowed the separation of solvent effects on pKa from kcat: delta pKa = pKD--pKH = 0.02--0.21, kH2O/kD2O = 1.20 +/- 0.09 in the direction of p-methoxybenzyl alcohol oxidation, and kH2O/kD2O = 0.50 +/- 0.05 and 0.58 +/- 0.06 for p-methoxybenzaldehyde reducation by NADH and [4-2H]NADH. The small effect of D2O on pKa, which contrasts with the common observation that delta pKa congruent to 0.4--0.6, is tentatively assigned to ionization of an active-site ZnOH2. The near absence of an isotope effect on kcat in the direction of alcohol oxidation rules out a mechanism involving concerted catalysis by an active-site base of hydride transfer. In the direction of aldehyde reduction, the observation of inverse isotope effects on kcat is concluded to reflect displacement of zinc-bound water by substrate to form an inner-sphere complex, subsequent to the E.S complex. Equilibrium alpha-secondary isotope effects, measured as a frame of reference for kinetic values, indicate KH/KT = 1.33 +/- 0.05 and 1.34 +/- 0.09 for the oxidation of [1(S)-3H]benzyl alcohol and p-methoxy[1(S)-3H]benzyl alcohol, respectively. Kinetic alpha-secondary isotope effects are within experimental error of equilibrium values, kH/kT = 1.34 +/- 0.07 and 1.38 +/- 0.02 for [1(S)-3H]benzyl alcohol and p-methoxy[1(S)-3H]benzyl alcohol oxidation, respectively. The near identity of kinetic and equilibrium alpha-secondary isotope effects in the direction of alcohol oxidation implicates a transition-state structure which resembles aldehyde with regard to bond hybridization properties. This result contrasts sharply with previously reported structure--reactivity correlations, which implicate a transition-state structure resembling alcohol with regard to charge properties. The significance of these findings to the mechanism of NAD(P)H-dependent redox reactions is discussed.