Transient oxidation as a mechanistic strategy in enzymatic catalysis

Enzymes that employ a transient oxidation mechanism catalyze transformations that are overall redox neutral, but involve intermediates that have a higher oxidation state than the substrates or products. An oxidation/reduction sequence may be used directly to promote isomerization reactions or indirectly to permit the formation of stabilized intermediates such as carbanions. This review will focus on three recent examples of nicotinamide-dependent enzymes that have been found to employ transient oxidation during catalysis: ADP-L-glycero-D-manno-heptose 6-epimerase, GDP-mannose 3,5-epimerase, and the 6-phosphoglucosidases from family 4. These enzymes are remarkable in their ability to catalyze either nonstereospecific hydride transfers or multiple chemical steps within a single active site.     

Importance of a serine proximal to the C(4a) and N(5) flavin atoms for hydride transfer in choline oxidase

Choline oxidase catalyzes the flavin-dependent, two-step oxidation of choline to glycine betaine with the formation of an aldehyde intermediate. In the first oxidation reaction, the alcohol substrate is initially activated to its alkoxide via proton abstraction. The substrate is oxidized via transfer of a hydride from the alkoxide α-carbon to the N(5) atom of the enzyme-bound flavin. In the wild-type enzyme, proton and hydride transfers are mechanistically and kinetically uncoupled. In this study, we have mutagenized an active site serine proximal to the C(4a) and N(5) atoms of the flavin and investigated the reactions of proton and hydride transfers by using substrate and solvent kinetic isotope effects. Replacement of Ser101 with threonine, alanine, cysteine, or valine resulted in biphasic traces in anaerobic reductions of the flavin with choline investigated in a stopped-flow spectrophotometer. Kinetic isotope effects established that the kinetic phases correspond to the proton and hydride transfer reactions catalyzed by the enzyme. Upon removal of Ser101, there is an at least 15-fold decrease in the rate constants for proton abstraction, irrespective of whether threonine, alanine, valine, or cysteine is present in the mutant enzyme. A logarithmic decrease spanning 4 orders of magnitude is seen in the rate constants for hydride transfer with increasing hydrophobicity of the side chain at position 101. This study shows that the hydrophilic character of a serine residue proximal to the C(4a) and N(5) flavin atoms is important for efficient hydride transfer.     

An inquiry into the source of stereospecificity of lactate dehydrogenase using substrate analogues and molecular modeling.

Lactate dehydrogenase catalyzes the stereospecific hydride transfer to and from the re face of the nicotinamide coenzyme. The demonstrated probability of transfer to the si face of less than 2 x 10(-8) indicates that the free energy of any diastereotopic transition state leading to a si transfer must be over 10 kcal/mol greater than the free energy for transfer to or from the re face. The general notion of closed, desolvated active sites suggests the a priori hypothesis that steric hindrance prevents the nicotinamide ring from assuming a conformation that would lead to transfer of the pro-S hydrogen. In this paper we report that the probability of transfer of the pro-S proton is less than 9 x 10(-7) with 3-pyridinealdehyde adenine dinucleotide as coenzyme and less than 4 x 10(-7) during the lactate dehydrogenase catalyzed disproportionation of glyoxylate. Examination of the crystal structure of lactate dehydrogenase further suggests that steric exclusion does not enforce the extreme stereospecificity of the reaction. An electrostatic interaction with the macrodipole associated with the alpha 2F helix is suggested as a potential molecular source of the stereospecificity.     

Function of coenzyme F420 in aerobic catabolism of 2,4, 6-trinitrophenol and 2,4-dinitrophenol by Nocardioides simplex FJ2-1A

2,4,6-Trinitrophenol (picric acid) and 2,4-dinitrophenol were readily biodegraded by the strain Nocardioides simplex FJ2-1A. Aerobic bacterial degradation of these pi-electron-deficient aromatic compounds is initiated by hydrogenation at the aromatic ring. A two-component enzyme system was identified which catalyzes hydride transfer to picric acid and 2,4-dinitrophenol. Enzymatic activity was dependent on NADPH and coenzyme F420. The latter could be replaced by an authentic preparation of coenzyme F420 from Methanobacterium thermoautotrophicum. One of the protein components functions as a NADPH-dependent F420 reductase. A second component is a hydride transferase which transfers hydride from reduced coenzyme F420 to the aromatic system of the nitrophenols. The N-terminal sequence of the F420 reductase showed high homology with an F420-dependent NADP reductase found in archaea. In contrast, no N-terminal similarity to any known protein was found for the hydride-transferring enzyme.     

Hydride transfer by dihydrofolate reductase. Causes and consequences of the wide range of rates exhibited by bacterial and vertebrate enzymes

Transient and steady-state kinetics have been examined for dihydrofolate reductase (DHFR) from a number of sources. Rates of hydride transfer at pH 7.65 cover a wide range, from 7 s-1 for DHFR from a strain of Lactobacillus casei (LCDHFR1) to 3000 s-1 for recombinant human DHFR (rHDHFR). In all cases as the pH is increased from 7 to 10, Vmax for the steady-state reaction decreases, and DVmax, the primary isotope effect, increases. This indicates a decrease in the rate of hydride transfer with increasing pH. The cross-over points, at which rates of product release and hydride transfer become equal, were calculated to occur at DVmax = 2.34. The higher the rate of hydride transfer at pH 7.65, the higher the pH of the cross-over point. For LCDHFR1 the low rate of hydride transfer results in this process being partially rate-limiting for the steady-state reaction even at pH 5, with a cross-over point at about pH 7. At pH 7.65 the burst phase associated with the initial conversion of enzyme-bound substrates to enzyme-bound products has an isotope effect of 3 or higher for LCDHFR and for DHFR from Escherichia coli (ECDHFR). In contrast, the vertebrate DHFRs (bovine, BDHFR; chicken, CDHFR; and rHDHFR) exhibit a burst of product formation which is only partially limited by hydride transfer at this pH (Dkb: 2.3, 2.2, and 2.1, respectively). An obligatory isomerization of the ternary substrate complex or of the ternary product complex is postulated to be partially rate-limiting for the vertebrate enzymes. At pH 5 LCDHFR1 and ECDHFR also exhibit evidence of such a rate-limiting obligatory conformational transition of the substrate or product ternary complex.     

Converting fructose to 5-hydroxymethylfurfural: a quantum mechanics/molecular mechanics study of the mechanism and energetics

We studied the energetics of the closed-ring mechanism of the acid-catalysed dehydration of d-fructose to 5-hydroxymethylfurfural (HMF) by carrying out canonical ensemble free-energy calculations using bias-sampling, hybrid Quantum Mechanics/Molecular Mechanics Molecular Dynamics simulations with explicit water solvent at 363 K. The quantum mechanical calculations are performed at the PM3 theory level. We find that the reaction proceeds via intramolecular proton and hydride transfers. Solvent dynamics effects are analysed, and we show that the activation energy for the hydride transfers is due to re-organization of the polar solvent environment. We also find that in some instances intramolecular proton transfer is facilitated by mediating water, whereas in others the presence of quantum mechanical water has no effect. From a micro-kinetic point of view, we find that the rate-determining step of the reaction involves a hydride transfer prior to the third dehydration step, requiring an activation free energy of 31.8 kcal/mol, and the respective rate is found in good agreement with reported experimental values in zeolites. Thermodynamically, the reaction is exothermic by .     

Effect of exogenous estrone sulfate on embryonic survival during asynchronous transfers in the pig

The effect of exogenous estrone sulfate (5 mg/day for 10 consecutive days starting on Day 10 after mating) on survival of embryos during asynchronous transfers was studied in Large White x Landrace gilts. Superinduction transfers were conducted by placing Day 4 embryos (younger) into mated Day-5 recipients (older) and vice versa. Treatment with estrone sulfate improved embryo survival in the transfer of younger embryos to recipients with a more developed uterine environment, but it did not affect the survival rate of older embryos in pregnant recipients. The results of the study also showed that when older embryos were transferred to a less developed uterine environment with or without estrone sulfate treatment they were better able to survine than younger embryos transferred to a more developed uterine environment.     

A twin-track approach has optimized proton and hydride transfer by dynamically coupled tunneling during the evolution of protochlorophyllide oxidoreductase

Protein dynamics are crucial for realizing the catalytic power of enzymes, but how enzymes have evolved to achieve catalysis is unknown. The light-activated enzyme protochlorophyllide oxidoreductase (POR) catalyzes sequential hydride and proton transfers in the photoexcited and ground states, respectively, and is an excellent system for relating the effects of motions to catalysis. Here, we have used the temperature dependence of isotope effects and solvent viscosity measurements to analyze the dynamics coupled to the hydride and proton transfer steps in three cyanobacterial PORs and a related plant enzyme. We have related the dynamic profiles of each enzyme to their evolutionary origin. Motions coupled to light-driven hydride transfer are conserved across all POR enzymes, but those linked to thermally activated proton transfer are variable. Cyanobacterial PORs require complex and solvent-coupled dynamic networks to optimize the proton donor-acceptor distance, but evolutionary pressures appear to have minimized such networks in plant PORs. POR from Gloeobacter violaceus has features of both the cyanobacterial and plant enzymes, suggesting that the dynamic properties have been optimized during the evolution of POR. We infer that the differing trajectories in optimizing a catalytic structure are related to the stringency of the chemistry catalyzed and define a functional adaptation in which active site chemistry is protected from the dynamic effects of distal mutations that might otherwise impact negatively on enzyme catalysis.     

Kinetic investigation of the functional role of phenylalanine-31 of recombinant human dihydrofolate reductase

The role of the active site residue phenylalanine-31 (Phe31) for recombinant human dihydrofolate reductase (rHDHFR) has been probed by comparing the kinetic behavior of wild-type enzyme (wt) with mutant in which Phe31 is replaced by leucine (F31L rHDHFR). At pH 7.65 the steady-state kcat is almost doubled, but the rate constant for hydride transfer is decreased to less than half that for wt enzyme, as is the rate of the obligatory isomerization of the substrate complex that precedes hydride transfer. Although steady-state measurements indicated that the mutation causes large increases in Km for both substrates, dissociation constants for many complexes are decreased. These apparent paradoxes are due to major mutation-induced decreases in rate constants (koff) for dissociation of folate, dihydrofolate, and tetrahydrofolate from all of their complexes. This results in a mechanism proceeding almost entirely by only one of the two pathways used by wt enzyme. Other consequences of these changes are a much altered dependence of steady-state kcat on pH, inhibition rather than activation by tetrahydrofolate, absence of hysteresis in transient-state kinetics, and a decrease in enzyme efficiency under physiological conditions. The results indicate that there is no quantitative correlation between dihydrofolate binding and the rate of hydride transfer for this enzyme.     

Mechanism of retrotransfer in conjugation: prior transfer of the conjugative plasmid is required.

Bacterial conjugation normally involves the unidirectional transfer of DNA from donor to recipient. Occasionally, conjugation results in the transfer of DNA from recipient to donor, a phenomenon known as retrotransfer. Two distinct models have been generally considered for the mechanism of retrotransfer. In the two-way conduction model, no transfer of the conjugative plasmid is required. The establishment of a single conjugation bridge between donor and recipient is sufficient for the transfer of DNA in both directions. In the one-way conduction model, transfer of the conjugative plasmid to the recipient is required to allow the synthesis of a new conjugation bridge for the transfer of DNA from recipient to donor. We have tested these models by the construction of a mutant of the self-transmissible, IncP plasmid RK2lac that allows the establishement of the conjugation bridge but is incapable of self-transfer. Four nucleotides of the nic region of the origin of transfer (oriT) were changed directly in the 67-kb plasmid RK2lac by a simple adaptation of the vector-mediated excision (VEX) strategy for precision mutagenesis of large plasmids (E. K.Ayres, V. J. Thomson, G. Merino, D. Balderes, and D. H. Figurski, J. Mol. Biol. 230:174-185, 1993). The resulting RK2lac oriT1 mutant plasmid mobilizes IncQ or IncP oriT+ plasmids efficiently but transfers itself at a frequency which is 10(4)-fold less than that of the wild type. Whereas the wild-type RK2lac oriT+ plasmid promotes the retrotransfer of an IncQ plasmid from Escherichia coli or Pseudomonas aeruginosa recipients, the RK2lac oriT1 mutant is severely defective in retrotransfer. Therefore, retrotransfer requires prior transfer of the conjugative plasmid to the recipient. The results prove that retrotransfer occurs by two sequential DNA transfer events.     

Metabolic characteristics of experimental free vascularized canine gracilis muscle transfers

A canine gracilis model was used to study muscle energy metabolism and enzyme activities after free vascularized muscle transfer. Fifteen male mongrel dogs underwent orthotopic, free transfer of the left gracilis with microneurovascular anastomosis. After a minimum of 10 months' recovery, muscle biopsy specimens were obtained from the transfers and the contralateral controls and analyzed for relative fiber type areas and maximum activities of phosphorylase, hexokinase, phosphofructokinase, glycerol-3-phosphate dehydrogenase (GPDH), pyruvate kinase, lactate dehydrogenase, citrate synthase, succinate dehydrogenase, 3-hydroxyacyl coenzyme A dehydrogenase (HAD), and creatine phosphokinase. Biopsy specimens obtained before and after a 10 minute, 20-Hz contraction were analyzed for glucose, glycogen, glycolytic intermediates, phosphocreatine, total creatine, and adenine nucleotides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, inosine monophosphate, and inosine). There was no significant transfer versus control difference in type I relative fiber area (45 +/- 4 percent versus 44 +/- 3 percent). Total creatine was significantly reduced in the transferred muscles relative to control (83.1 +/- 3.0 mmol/kg versus 100.6 +/- 5.1 mmol/kg dry weight). Maximal activities of phosphorylase, pyruvate kinase, lactate dehydrogenase, citrate synthase, succinate dehydrogenase, HAD, and creatine phosphokinase were diminished in transfers relative to controls, although hexokinase activity was significantly higher in the freely transferred gracilis muscles. During the 20-Hz contraction, muscle transfers produced less force initially, although the force/time integral over the 10-minute stimulation was similar in transfers (277 +/- 25 N/g/second) and controls (272 +/- 24 N/g/second). The contraction was associated with significant glvcogen use and lactate accumulation in both transfers and controls, although this was less pronounced for the transfers. Glycolytic flux appeared muted in the transfers relative to controls. Significant, similar high-energy phosphagen reductions and inosine monophosphate accumulation were noted during the contraction in both groups. Contractile activity is associated with the expected pattern of muscle metabolite changes following free vascularized transfer, indicating the components of cellular energy metabolism are not qualitatively altered after microneurovascular muscle transfer. In contrast, quantitative differences suggest that free vascularized muscle transfer can be associated with a muscle enzyme profile consistent with deconditioning and the presence of denervated muscles fibers in the absence of fiber type profile changes.     

Determination of the redox potentials and electron transfer properties of the FAD- and FMN-binding domains of the human oxidoreductase NR1.

Human novel reductase 1 (NR1) is an NADPH dependent diflavin oxidoreductase related to cytochrome P450 reductase (CPR). The FAD/NADPH- and FMN-binding domains of NR1 have been expressed and purified and their redox properties studied by stopped-flow and steady-state kinetic methods, and by potentiometry. The midpoint reduction potentials of the oxidized/semiquinone (-315 +/- 5 mV) and semiquinone/dihydroquinone (-365 +/- 15 mV) couples of the FAD/NADPH domain are similar to those for the FAD/NADPH domain of human CPR, but the rate of hydride transfer from NADPH to the FAD/NADPH domain of NR1 is approximately 200-fold slower. Hydride transfer is rate-limiting in steady-state reactions of the FAD/NADPH domain with artificial redox acceptors. Stopped-flow studies indicate that hydride transfer from the FAD/NADPH domain of NR1 to NADP+ is faster than hydride transfer in the physiological direction (NADPH to FAD), consistent with the measured reduction potentials of the FAD couples [midpoint potential for FAD redox couples is -340 mV, cf-320 mV for NAD(P)H]. The midpoint reduction potentials for the flavin couples in the FMN domain are -146 +/- 5 mV (oxidized/semiquinone) and -305 +/- 5 mV (semiquinone/dihydroquinone). The FMN oxidized/semiquinone couple indicates stabilization of the FMN semiquinone, consistent with (a) a need to transfer electrons from the FAD/NADPH domain to the FMN domain, and (b) the thermodynamic properties of the FMN domain in CPR and nitric oxide synthase. Despite overall structural resemblance of NR1 and CPR, our studies reveal thermodynamic similarities but major kinetic differences in the electron transfer reactions catalysed by the flavin-binding domains.     

Laparoscopic transfer of ovine and cervine embryos using the transpic technique

A transpic technique was developed to transfer embryos to 352 sheep and 4 deer recipients using a laparoscope, a modified pair of Allis forceps and a modified Cassou aspic normally used for laparoscopic uterine insemination. The overall proportion of uncomplicated transfers in Experiment 1 in 216 recipient ewes was 90.7% (range between groups 80 to 100%), 3.7% of the transfers were presumed to be loss of embryos during expulsion from the transpic, and 5.6% were apparent transfers into the uterine wall. In Experiment 2,83% of transfers into 136 ewe recipients were uncomplicated, 5% were presumed to be loss of embryos during expulsion, 1% was apparent transfer into the uterine wall, and 11% involved 2 attempts at transfer. Only 34% of 116 recipients receiving low-quality frozen-thawed embryos were pregnant and 24% of the 226 embryos survived to term. In contrast, high pregnancy rates (>80%) and embryo survival rates (>70%) were achieved following uncomplicated and twice attempted transfers of fresh embryos. Pregnancy rates and embryo survival rates were low (<2%) following the presumed loss of embryos during expulsion and apparent transfers into the uterine wall. All 4 deer transfers were uncomplicated and 2 2 good-quality embryos survived to term compared with 0 2 low-medium quality embryos. The transpic technique is a moderately invasive technique which permits fast (15 to 20/h) and reliable transfer of embryos in small ruminants. With appropriate care, nearly all of the embryos can be correctly placed in the uterus, and high pregnancy rates and embryo survival rates can be achieved using this technique.     

An internal equilibrium preorganizes the enzyme-substrate complex for hydride tunneling in choline oxidase

The hydride transfer reaction catalyzed by choline oxidase under irreversible regime, i.e., at saturating oxygen, was shown in a recent study to occur quantum mechanically within a highly preorganized active site, with the reactive configuration for hydride tunneling being minimally affected by environmental vibrations of the reaction coordinate other than those affecting the distance between the alpha-carbon of the choline alkoxide substrate and the N(5) atom of the enzyme-bound flavin cofactor [Fan, F., and Gadda, G. (2005) J. Am. Chem. Soc. 127, 17954-17961]. In this study, we have determined the effects of pH and temperature on the substrate kinetic isotope effects with 1,2-[2H4]choline as substrate for choline oxidase at 0.2 mM oxygen to gain insights on the mechanism of hydride transfer under reversible catalytic regime. The data presented indicated that the kinetic complexity arising from the net flux through the reverse of the hydride transfer step changed with temperature, with the hydride transfer reaction becoming more reversible with increasing temperatures. After this kinetic complexity was accounted for, analyses of the kcat/Km and D(kcat/Km) values determined at 0.2 mM according to the Eyring and Arrhenius formalisms suggested that the quantum mechanical nature of the hydride transfer reaction is, not surprisingly, maintained during enzymatic catalysis under reversible regime. A comparison of the thermodynamic and kinetic parameters of the hydride transfer reaction under reversible and irreversible catalytic regimes showed that the enthalpies of activation (DeltaH++) were significantly larger in the reversible catalytic regime. This reflects the presence of an enthalpically unfavorable internal equilibrium of the enzyme-substrate Michaelis complex occurring prior to, and independently from, CH bond cleavage. Such an internal equilibrium is required to preorganize the enzyme-substrate complex for efficient quantum mechanical tunneling of the hydride ion from the substrate alpha-carbon to the flavin N(5) atom.     

Transient kinetic and deuterium isotope effect studies on the catalytic mechanism of phosphoglycerate dehydrogenase.

The catalytic mechanism of the phosphoglycerate dehydrogenase reaction in both directions was investigated by studying: (a) pre-steady state transients in reduced coenzyme appearance or disappearance or disappearance and in protein fluorescence; (b) deuterium isotope effects on the transients and on the steady state reactions; and (c) the partial reaction between the enzyme-NADH complex and hydroxypyruvate-P. These studies led to the scheme below for the ternary complex interconversion. E1-NADH-hydroxypyruvate-P(1)equilibriumE2-NADH-hydroxypyruvate-P(2)equilibriumE3-NADH-hydroxypyruvate-P + H+(3)equilibriumE3-NAD+-3-phosphoglycerate(4)equilibriumE4-NAD+-3-phosphoglycerate Steps 1,2, and 4 are ternary complex isomerizations. Step 3 is the hydride transfer. Under steady state conditions isomerization 2 is the rate-determining step in the direction of hydroxypyruvate-P reduction at higher pH values. At lower pH values, the hydride transfer step is also partially rate-determining. The rate-determining step in the direction of 3-phosphoglycerate oxidation occurs subsequent to the hydride transfer step at higher pH values. At lower pH values the rate is determined by both isomerization 4 and the hydride transfer step. Isomerizations 1, 2, and 4 were inhibited by serine, an allosteric inhibitor, indicating that the inactive conformation of the enzyme is incapable of performing any of the steps of the ternary complex interconversion. Phosphoglycerate dehydrogenase corresponds to a V-type allosteric enzyme. When the enzyme-NADH complex was mixed with hydroxypyruvate-P at pH 8.5, a rapid quenching of enzymebound NADH fluorescence occurred. This process was studied under pseudo-first order conditions and shown to be the result of hydroxypyruvate-P binding.     

Kinetic and chemical mechanism of the dihydrofolate reductase from Mycobacterium tuberculosis

Dihydrofolate reductase from Mycobacterium tuberculosis (MtDHFR) catalyzes the NAD(P)-dependent reduction of dihydrofolate, yielding NAD(P)(+) and tetrahydrofolate, the primary one-carbon unit carrier in biology. Tetrahydrofolate needs to be recycled so that reactions involved in dTMP synthesis and purine metabolism are maintained. In this work, we report the kinetic characterization of the MtDHFR. This enzyme has a sequential steady-state random kinetic mechanism, probably with a preferred pathway with NADPH binding first. A pK(a) value for an enzymic acid of approximately 7.0 was identified from the pH dependence of V, and the analysis of the primary kinetic isotope effects revealed that the hydride transfer step is at least partly rate-limiting throughout the pH range analyzed. Additionally, solvent and multiple kinetic isotope effects were determined and analyzed, and equilibrium isotope effects were measured on the equilibrium constant. (D(2)O)V and (D(2)O)V/K([4R-4-(2)H]NADH) were slightly inverse at pH 6.0, and inverse values for (D(2)O)V([4R-4-(2)H]NADH) and (D(2)O)V/K([4R-4-(2)H]NADH) suggested that a pre-equilibrium protonation is occurring before the hydride transfer step, indicating a stepwise mechanism for proton and hydride transfer. The same value was obtained for (D)k(H) at pH 5.5 and 7.5, reaffirming the rate-limiting nature of the hydride transfer step. A chemical mechanism is proposed on the basis of the results obtained here.     

Quantitation of Beta Adrenergic Receptors in Rat Liver: Confounding Effect of Displaceable But Nonstereospecific Antagonist Binding

Abstract

In rat liver membranes three types of ligand binding were seen using [³H]-dihydroalprenolol (DHA) and [¹²⁵I]-hydroxybenzylpindolol (HYP): binding stereospecifically displaced by β-adrenergic agonists and antagonists, binding nonstereospecifically displaced by β-adrenergic antagonists, and binding which was not displaced by β-adrenergic agonists or antagonists.

The magnitude of the nonstereospecific displaceable binding varied with the physiological state of the animal. It was sufficient to prevent the quantitation of the stereospecific displaceable binding in some preparations from young rats but in all preparations of rats greater than 150 g or more than about 6 weeks of age. In adrenalectomized weanling rats 10–30% of the total binding was of nonstereospecific displaceable type while in control rats it comprised up to 60% of the total binding. Addition of 5 X 10^-6 M phentolamine to the assay eliminated a large proportion of the nonstereospecific displaceable binding. When phentolamine was included in the assay, liver membranes from weanling rats stereo-specifically bound 30–35% of total binding; membranes from adrenalectomized rats showed stereospecific binding of up to 50 to 80%.

Because the amount of displaceable, nonstereospecific binding varied greatly depending on the physiologic state of the animals, stereospecific displacement should be monitored for every type of liver membrane preparation. Furthermore, animal age is an important variable. Using the published antagonist binding methodology (DHA or HYP) in liver membranes, it is not presently possible to quantitate liver β-adrenergic receptors in normal rats that have reached maturity.     

Direct measurement of the pKa of aspartic acid 26 in Lactobacillus casei dihydrofolate reductase: implications for the catalytic mechanism.

The ionization state of aspartate 26 in Lactobacillus casei dihydrofolate reductase has been investigated by selectively labeling the enzyme with [13Cgamma] aspartic acid and measuring the 13C chemical shifts in the apo, folate-enzyme, and dihydrofolate-enzyme complexes. Our results indicate that no aspartate residue has a pKa greater than approximately 4.8 in any of the three complexes studied. The resonance of aspartate 26 in the dihydrofolate-enzyme complex has been assigned by site-directed mutagenesis; aspartate 26 is found to have a pKa value of less than 4 in this complex. Such a low pKa value makes it most unlikely that the ionization of this residue is responsible for the observed pH profile of hydride ion transfer [apparent pKa = 6.0; Andrews, J., Fierke, C. A., Birdsall, B., Ostler, G., Feeney, J., Roberts, G. C. K., and Benkovic, S. J. (1989) Biochemistry 28, 5743-5750]. Furthermore, the downfield chemical shift of the Asp 26 (13)Cgamma resonance in the dihydrofolate-enzyme complex provides experimental evidence that the pteridine ring of dihydrofolate is polarized when bound to the enzyme. We propose that this polarization of dihydrofolate acts as the driving force for protonation of the electron-rich O4 atom which occurs in the presence of NADPH. After this protonation of the substrate, a network of hydrogen bonds between O4, N5 and a bound water molecule facilitates transfer of the proton to N5 and transfer of a hydride ion from NADPH to the C6 atom to complete the reduction process.     

Effect of pH on the liver alcohol dehydrogenase reaction.

New transient kinetic methods, which allow kinetics to be carried out under conditions of excess substrate, have been employed to investigate the kinetics of hydride transfer from NADH to aromatic aldehydes and from aromatic alcohols to NAD+ as a function of pH. The hydride transfer rate from 4-deuterio-NADH to beta-naphthaldehyde is nearly pH independent from pH 6.0 to pH 9.9; the isotope effect is also pH independent with kappa-H/kappaD congruent to 2.3. Likewise, the rate of oxidation of benzyl alcohol by NAD+ changes little with pH between pH 8.75 and pH 5.9; the isotope effect for this process is between 3.0 and 4.4. Earlier substituent effect studies on the reduction of aromatic aldehydes were consistent with electrophilic catalysis by either zinc or a protonic acid. The pH independence of hydride transfer is consistent with electrophilic catalysis by zinc since such catalysis by protonic acid (with a pK between 6.0 and 10.0) would show strong pH dependence. However, protonic acid catalysis cannot be excluded if the pKa of the acid catalyst in the ternary NADH-E-RCOH complex were smaller than 6.0 or smaller than 10.0. The two kinetic parameters changing significantly with pH are the kinetic binding constant for ternary complex formation with aromatic alcohol and the rate of dissociation of aromatic alcohols from enzyme. This is consistent with base-catalyzed removal of a proton from alcohol substrated and consequent acid catalysis of protonation of a zinc-alcoholate complex. The equilibrium constant for hydride transfer from benzaldehyde to benzyl alcohol at pH 8.75 is K-eq equals kappa-H/kappa-H equals 42; this constant has important consequences concerning subunit interactions during liver alcohol dehydrogenase catalysis.     

Effects of apolipoprotein structure on the kinetics of apolipoprotein transfer between phospholipid vesicles

The kinetics and mechanism of transfer of 14C-labeled human apolipoproteins A-I, A-II and C-III1 between small unilamellar vesicles (SUV) have been investigated. Ion exchange chromatography was used for rapid separation of negatively charged egg phosphatidylcholine (PC)/dicetyl phosphate donor SUV containing bound 14C-labeled apoprotein from neutral egg PC acceptor SUV present in 10-fold molar excess. The transfer kinetics of these apolipoproteins at 37 degrees C are consistent with the existence of fast, slow and apparently 'nontransferrable' pools of SUV-associated lipoprotein: the transfers from these pools occur on timescales of seconds (or less), minutes/hours and days/weeks, respectively. For donor SUV containing about 15 apoprotein molecules per vesicle and at a donor SUV concentration of 0.15 mg phospholipid/ml incubation mixture, the sizes of the fast kinetic pools for apolipoproteins A-I, A-II and C-III1 associated with donor SUV are 2, 10 and 11%, respectively. The sizes of the slow kinetic pools for these apolipoproteins are 16, 71 and 50%, respectively. The transfer of the various apolipoproteins from the slow kinetic pool follows first order kinetics and the half-time (t1/2) values are in the order: apo C-III1 less than apo A-I. Increasing the number of apoprotein molecules per donor SUV enlarges the size of the fast pool and increases the t1/2 of slow transfer. The differences in the kinetics of apolipoprotein transfer between SUV are consequences of the variations in the primary and secondary structures of the apolipoprotein molecules. The slow transfer of apoprotein molecules is mediated by collisions between donor and acceptor SUV; the rate is dependent on the apoprotein molecular weight with larger molecules transferring more slowly from donor SUV containing the same lipid/protein molar ratio. The hydrophobicity of the apoprotein molecule is also significant with less hydrophobic molecules transferring more rapidly. Further understanding of the differences in the kinetics of transfer of these apolipoproteins will require more knowledge of their secondary and tertiary structures.