Energetics of proline racemase: double fractionation experiment, a test for concertedness and for transition-state dominance

To test whether a reaction involving the making and/or breaking of two bonds at two sites is concerted (and proceeds through a single transition state) or is stepwise (and involves a reaction intermediate in which only one bond has been made or broken), we have measured the isotopic fractionation at one site as a function of isotopic substitution at the other site. In the case of proline racemase, the discrimination against solvent deuterium in the product when the reaction is run in mixed H2O-D2O is measured for the reaction both of [2-1H]proline and of [2-2H]proline. The isotopic fractionation at the solvent site may in principle be smaller, the same, or larger, when the 2H-labeled substrate is used rather than the 1H substrate, and--depending upon the nature of the catalyzing groups--this information indicates whether the reaction is stepwise, or concerted, or whether an isotopically insensitive transition state is partially rate determining. Experimentally, we have found that the discrimination against solvent deuterium in the product L-proline is the same, whether D-[2-1H]proline or D-[2-2H]proline is the substrate. This result requires that the substrate and product "on-off" steps are faster than the racemization step and that the racemization reaction proceeds either in a concerted manner or in a stepwise fashion involving enzyme catalytic groups (e.g., thiols) having ground-state fractionation factors around 0.5.     

Energetics of proline racemase: fractionation factors for the essential catalytic groups in the enzyme-substrate complexes

The fractionation factors of protons bound to the essential catalytic groups in proline racemase have been determined by comparison of the time courses of two competitive deuterium washout experiments. The rate of achievement of the maximum perturbation in the optical rotation has been measured in the oversaturated region (that is, at high substrate concentrations) under two conditions: in the first, we start with an equimolar mixture of deuterated substrate S' and of unlabeled product P; in the second, we again start with equal concentrations of substrate and product, but the concentration of the deuterated material S' is less than 20% that of S. The different concentrations of deuterated substrate produce different levels of deuteration of the enzyme's catalytic groups, the kinetic consequence of which allow the fractionation factors of these enzymic groups to be determined. The observed values for the fractionation factors of the enzyme's groups of 0.55 +/- 0.1 are only consistent with these groups' being thiols. This conclusion is supported by results of measurements of the solvent isotope effect determined in the unsaturated regime. These findings confirm the earlier suggestion of Abeles and his group that two cysteine residues mediate the catalysis of proline racemization by this enzyme.     

On the structures of flavoprotein D-amino acid oxidase purple intermediates. A resonance Raman study

Resonance Raman (RR) spectra were obtained in H2O or D2O solution for the purple intermediates of D-amino acid oxidase (DAO) with isotopically labeled substrates, i.e., [1-13C]-, [2-13C]-, [3-13C]-, [15N]-, and [3,3,3-D3]alanine; [carboxyl-13C]- and [15N]proline. RR spectra were also measured for the intermediates of DAO reconstituted with isotopically labeled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]FAD in D2O. The isotopic shift of the 1692 cm-1 band upon [15N]- or [2-13C]-substitution of alanine indicates that the band is due to the C = N stretching mode of an imino acid derived from D-alanine, i.e., alpha-iminopropionate. The 1658 cm-1 band with D-proline was also assigned to the C = N stretching mode of an imino acid derived from D-proline, i.e., delta 1-pyrrolidine-2-carboxylate, since the band shifts to 1633 cm-1 upon [15N]-substitution and its stretching frequency is generally found in this frequency region. Since the band shifts to low frequency in D2O, the imino acid should have a protonated imino group such as the C = N+1H form. The intense band at 1363 cm-1 with D-alanine was assigned to a mixing of the CO2- symmetric stretching and CH3 symmetric deformation modes in alpha-iminopropionate, based on the isotope effects. The 1359 cm-1 band with D-proline has probably contributions of CO2- symmetric stretching and CH2 wagging, considering the isotope effects with [carboxyl-13C]proline. The 1359 cm-1 band with D-proline was split into 1371 cm-1 and 1334 cm-1 bands in D2O. As this splitting of the 1359 cm-1 band with D-proline in D2O can not be interpreted only by the replacement of the C = N+1-H proton by deuterium, the carboxylate of the imino acid probably interacts with the enzyme through some proton(s) exchangeable by deuterium(s) in D2O. The bands around 1605 cm-1 which shift upon [4a-13C]- and [4,10a-13C2]-labeling of FAD are derived from a fully reduced flavin, because the isotopic shifts of the band are very different from those of the bands of oxidized or semiquinoid flavin observed near 1605 cm-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence that the nucleic acid base queuine is incorporated intact into tRNA by animal cells

Queuine (the base of queuosine, Q) catalytically reduced with tritium or deuterium yields a derivative in which the proton at C-8 (purine numbering system) has been exchanged and the cyclopentene ring has been reduced to a cyclopentane ring. Mouse fibroblast tRNA has been labeled by culturing the cells in medium supplemented with [3H]- and [2H]dihydroqueuine. Such tRNA yields, upon hydrolysis, the nucleoside dihydroqueuosine and a saccharide derivative of dihydroqueuosine. Each product has been identified unambiguously by mass spectrometry and chromatography. Both the 3H- and 2H-labeled material coeluted, and no unlabeled Q nucleoside was found. Therefore, dihydroqueuine is incorporated intact into tRNA in mammalian cells. Furthermore, fractionation of the labeled tRNA on concanavalin A-agarose, which specifically binds the mannosyl-Q-containing tRNAAsp, has shown that the dihydroqueuosine-containing tRNAAsp is mannosylated. This is the first direct evidence that queuine is incorporated intact into mammalian tRNA in vivo.     

Gamma-butyrobetaine hydroxylase: stereochemical course of the hydroxylation reaction

The stereochemical course of the aliphatic hydroxylation of gamma-butyrobetaine by calf liver and by Pseudomonas sp AK1 gamma-butyrobetaine hydroxylases has been determined. With [3(RS)-3-3H]-gamma-butyrobetaine or [3(R)-3-3H]-gamma-butyrobetaine as substrate, a rapid and significant loss of tritium to the medium occurred. On the other hand, with [3(S)-3-3H]-gamma-butyrobetaine, only a negligible release of tritium to the aqueous medium was observed. Indeed, on hydroxylation of [3(S)-3-2H]-gamma-butyrobetaine by either the calf liver or bacterial hydroxylase, the isolated product L-carnitine was found to have retained all of the deuterium initially present in the 3(S) position. Since the absolute configuration of the product L-carnitine has been determined to be R, such results are only compatible with a hydroxylation reaction that proceeded with retention of configuration. With [methyl-14C,3(R)-3-3H]-gamma-butyrobetaine as substrate for the calf liver hydroxylase, the percentage of tritium retained in the [methyl-14C]-L-carnitine product was determined as a function of percent reaction. The results of these studies indicated that pro-R hydrogen atom abstraction exceeded 99.9%. Experiments using racemic [methyl-14C,3(RS)-3-3H]-gamma-butyrobetaine as substrate yielded similar results and additionally allowed us to estimate alpha-secondary tritium kinetic isotope effects of 1.10 and 1.31 for the bacterial and calf liver enzymes, respectively. These results are discussed within the context of the radical mechanism for gamma-butyrobetaine hydroxylase previously proposed [Blanchard, J. S., & Englard, S. (1983) Biochemistry 22, 5922], and the required topographical arrangement of enzymic oxidant and substrate is illustrated.     

Reaction mechanism and structure of the active site of proline racemase.

Proline racemase catalyzes the interconversion of D- and L-proline. Previous studies in this laboratory have established that the reaction proceeds by means of a two-base mechanism in which one base on the enzyme removes the substrate alpha-hydrogen as a proton and the conjugate acid of another base donates a proton to the opposite side of the alpha-carbon (Cardinale, G.J., and Abeles, R.H., (1968), Biochemistry 7, 3970. An assumption of the proposed mechanism was that no proton exchange occurs from the enzyme-substrate complex. In the present study, we have shown that the rate of 3H release from DL-[alpha-3H]proline, in the presence of proline racemase, decreases with increasing proline concentrations. These results establish that release of the substrate derived proton from the enzyme occurs largely, possibly exclusively, after release of the product. Under initial velocity conditions, the rate of 3H release from L-[alpha-3H]proline is not reduced with increasing L-proline concentrations. Thus, the enzyme-bound proton derived from one isomer can only be "captured" by the other isomer. We conclude that there are two forms of the enzyme; one binds L-proline and the other D-proline. Release of the substrate derived proton from enzyme is more rapid than the interconversion of these two forms. These results are consistent with the previously proposed mechanism. Proline racemase is composed of similar subunits of mol wt 38,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. Equilibrium dialysis experiments detect only one substrate binding site for every two subunits. When the oxidized form of the enzyme, which is inactive and cannot bind substrate, is reduced by thiol to yield active enzyme, two cysteine sulfhydryl groups per dimer become available to react with iodoacetate. Inactivation of the enzyme occurs upon modification of one of these cysteines. All iodoacetate incorporation occurs at the same point in the primary sequence of the enzyme, and can be prevented by the presence of proline or pyrrole-2-carboxylate, a substrate analog. A model is proposed in which a single active site is formed by elements of two identical subunits. Although the data are consistent with this model, another interpretation, in which half of the subunits are nonfunctional, cannot be ruled out.     

Serendipitous discovery of a pH-dependant atropisomer bond rotation: toward a write-protectable chiral molecular switch?

Owing to slow rotation of a sterically constrained dimethylamide substituent, two slowly interconverting enantiomers of a preclinical candidate for pharmaceutical development, 1, (6-(3-Chloro-4-fluoro-benzyl)-4-hydroxy-2-methyl-3,5-dioxo-2,3,5,6,7,8-hexahydro-[2,6]naphthyridine-1-carboxylic acid dimethylamide) are observed by chiral chromatography. Isolation of pure enantiomer by preparative chiral chromatography followed by enantiopurity analysis over time allowed for a study of the kinetics of enantiomer interconversion under a variety of conditions. Relatively slow racemization was observed in alcohol solvents, with a half life on the order of 5-10 h. A dramatic influence of aqueous buffer pH on racemization was noted, with higher pH leading to rapid racemization within a few minutes, and lower pH leading to essentially no racemization for periods up to a week. A hypothesis explaining this unusual effect of pH on carboxamide bond rotation is offered, and some suggestions for potential utility of such a system are considered.     

Synthesis, biological activity, and tritiation of L-3, 4-dehydroproline-containing peptides

A series of analogs of thyroliberin (TRH) ([L-delta3Pro3]-TRH, [D-delta3Pro3]-TRH, [L-3-MeHis2, L-delta3Pro3]-TRH) in which proline was replaced by L- or D-3, 4-dehydroproline was synthesized. The analogs exhibited approximately the same biological activity as the corresponding proline-containing peptides. These analogs and others in which 3, 4-dehydroproline is present at the NH2-terminal, COOH-terminal or internal positions in the peptide were successfully reduced with deuterium or tritium to provide the 3, 4-2H2-proline or 3,4-3H2-proline analogs, respectively, with near theoretical values of substitution. A novel procedure for the chemical resolution of DL-3, 4-dehydroproline is also described.     

Comparison of the function of L- and D-proline as compatible solute inEscherichia coli K-12 under high osmolarity

We compared the function of L- and D-proline as compatible solute inEscherichia coli K-12 cells under high osmolarity. Growth ofE. coli K-12 in a Davis minimal medium was inhibited at 0.5 M and 1 M NaCl, but it was recovered by the addition of L-proline. Glucose uptake was reduced with increase of external NaCl concentrations, but it was improved by the addition of L-proline. On the other hand, the addition of D-proline did not show the role of compatible solute although accumulated in cells. On the analysis ofE. coli proline transporter mutants, difference of the affinity of proline transporters for D-proline was observed at PutP and ProP. These results presumed that the functional disorder of D-proline as compatible solute was caused by its structural feature in cells.     

The consequences of the isotope effect on proline dehydrogenation rates estimated by the tritium loss method.

Loss of tritium from a substrate is often used to estimate the rate of dehydrogenation. However, loss of 3H may be much slower than loss of H because of the tritium isotope effect. In order to assess the impact of the tritium isotope effect, loss of 3H from the C-5 position of proline during dehydrogenation by rat liver mitochondria and bacteroids from soybean (Glycine max [L.] Merrill) nodules was compared with appearance of 14C in products of [14C]proline dehydrogenation. Incubations were carried out in the presence of o-aminobenzaldehyde (added to trap the initial product, delta 1-pyroline-5-carboxylate). The fraction of total 14C products trapped by o-aminobenzaldehyde varied from 0.07 to 0.75 depending upon experimental conditions. With rat liver mitochondria, dehydrogenation of [14C]proline was between 3.27 and 9.25 times faster than dehydrogenation of 3H proline, depending upon assay conditions. Soybean nodule bacteriods dehydrogenated [14C]proline about 5 times faster than [3H]proline. We conclude the following: (i) the rate of proline dehydrogenation may be greatly underestimated by the tritium assay because of the tritium isotope effect, and (ii) the 14C assay may underestimate the rate of proline dehydrogenation if it is assumed that o-aminobenzaldehyde quantitatively traps delta 1-pyrroline-5-carboxylate under all conditions. The simplicity of the tritium assay makes it attractive for routine use. However, its use requires determination of the tritium isotope effect, under the specific conditions of the assay, in order to correct the results. The considerations discussed here have broad applicability to any dehydrogenase assay employing tritium loss.     

Enzymic generation of chiral acetates. A quantitative evaluation of their configurational assay.

1. R-Acetate was generated enzymically from R-acetate in the sequence acetate leads to malate leads to oxaloacetate leads to acetate, and S-acetate likewise from S-acetate. It was concluded that the formation of malate on malate synthase involves the operation of a normal isotopic effect combined with inversion of configuration. The malate synthase kH/k2H was determined as 3.7 +/- 0.5 by a method which yields results independently of the stereochemical purity of the chiral acetates used initially. 2. R-Acetate was also generated from R-acetate in the sequence acetate leads to citrate leads to malate leads to oxaloacetate leads to acetate, and S-acetate likewise from S-acetate. The conclusion is the same as given above, but refers to the formation of citrate on the re-synthase. 3. 2S,3R-[2-2H1,3-2H1,3H1]Malate and 2S,3S-[2-2H1,3-2H1]malate were prepared from 2S-[2,3-2H3]malate by treatment with fumarase in tritiated water and normal water, respectively. It was assumed that these malate specimens were pure with respect to chirality as generated by isotopic labelling. 4. These two malate specimens were partially converted (about 9%) to acetates in conditions where no racemization at the level of transiently formed oxaloacetate occurred. That no racemization took place was demonstrated experimentally. Oxidative enzymic hydrolysis of 2S,3R-[2-2H1,3-2H1,3H1]malate in normal water and of 2S,3S-[2-2H1,3-2H1]malate in tritiated water produced S-[2H1,3H1]acetate and R-[2H1,3H1]acetate, respectively. 5. The isolated R-[2H1,3H1]acetate and S-[2H1,3H1]acetate on configurational analysis yielded malates which in the presence of fumarase retained 79.7 +/- 0.7% and 20.3 +/- 0.9%, respectively, of their total tritium content. The symmetric deviation from the 50% value found with [3H1]acetate strengthens the conclusion that stereochemically pure chiral acetates were analyzed. The malate synthase kH/k2H was determined from the data of this study as 3.9 +/- 0.2. 6. The average of the values given under paragraphs 1 and 5 for the isotopic discrimination on malate synthase corresponds to kH/k2H=3.8 +/- 0.1. It was concluded that the configurational analysis of stereochemically pure R-[2H1,3H1]acetate and S-[2H1,3H1]acetate yields malates which in the presence of fumarase retain 79 +/- 2% and 21 +/- 2%, respectively, of their total tritium content. Hence, a deviation of 29 +/- 2% from the 50% value represents the actual amplitude of the configurational assay. 7. Outlines are given for an enzymic generation of chiral acetates in preparative scale.     

Use of multiple isotope effects to study the mechanism of 6-phosphogluconate dehydrogenase

The multiple isotope effect method of Hermes et al. [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106-5114] has been used to study the mechanism of the oxidative decarboxylation catalyzed by 6-phosphogluconate dehydrogenase from yeast. 13C kinetic isotope effects of 1.0096 and 1.0081 with unlabeled or 3-deuterated 6-phosphogluconate, plus a 13C equilibrium isotope effect of 0.996 and a deuterium isotope effect on V/K of 1.54, show that the chemical reaction after the substrates have bound is stepwise, with hydride transfer preceding decarboxylation. The kinetic mechanism of substrate addition is random at pH 8, since the deuterium isotope effect is the same when either NADP or 6-phosphogluconate or 6-phosphogluconate-3-d is varied at fixed saturating levels of the other substrate. Deuterium isotope effects on V and V/K decrease toward unity at high pH at the same time that V and V/K are decreasing, suggesting that proton removal from the 3-hydroxyl may precede dehydrogenation. Comparison of the tritium effect of 2.05 with the other measured isotope effects gives limits of 3-4 on the intrinsic deuterium and of 1.01-1.05 for the intrinsic 13C isotope effect for C-C bond breakage in the forward direction and suggests that reverse hydride transfer is 1-4 times faster than decarboxylation.     

Study, by an enzymatic method, of the isomers of L-proline (3H-4) and of the effects of preservation]

By use of procollagen proline hydroxylas which catalyses the substitution of the hydrogen in the trans position on carbon 4 with an hydroxyl on L-proline residues of procollagen, we have shown that the L-[3H-4] proline product of C.E.N. Saclay contained 29 per cent of the trans and 71 per cent of the cis form. It was also possible to verify the molecular stability of the isomers and that a small amount of tritium was randomly linked to the whole molecule.     

Biosynthesis of trans-4-hydroxy-L-proline by Streptomyces griseoviridus.

Radioisotopic experiments have revealed that free trans-4-hydroxy-L-proline is an intermediate synthesized from L-proline during formation of the peptide-bound cis-4-hydroxy-D-proline residue in the antibiotic, etamycin. This conclusion was based on the fact that 1) both radiolabeled L-proline and trans-4-hydroxy-L-proline are precursors of the bound D-imino acid as noted previously by Hook and Vining ((1973) J. Chem. Soc. Chem. Commun. 185-186; (1973) Can. J. Biochem. 51, 1630-1637), 2) the unlabeled trans isomer specifically inhibited the incorporation of radiolabel from proline into the antibiotic, 3) the 14C-hydroxyimino-acid was isolated from the intracellular pool and medium following incubations with L-[14C]proline during antibiotic biosynthesis and when etamycin synthesis was blocked by D-leucine. By means of chromatographic and enzymatic analyses, it was established that the free imino acid possesses the trans-L configuration.     

Stereochemistry of propionyl-coenzyme A and pyruvate carboxylations catalyzed by transcarboxylase.

The stereochemistry of the two half-reactions catalyzed by the biotin-containing enzyme, transcarboxy-lase from Propionobacteria shermanii, has been determined. The pro-R hydrogen at C-2 of propionyl-coenzyme A is replaced by CO2 in formation of the S isomer of methylmalonyl-CoA, defining the process as retention of configuration. This C-2 hydrogen is abstracted at a rate identical with product formation. For the other half-reaction, pyruvate to oxalacetate, the chiral methyl group methodology of Rose (I. A. Rose (1970), J. Biol. Chem. 245, 6052) was employed. First, it was determined with [3-2-He]pyruvate that a kinetic deuterium isotope effect of 2.1 occurs at Vmax in this carboxyl transfer, indicating that the necessary requirement for discrimination against heavy isotopes of hydrogen existed. Then, 3(S)-[3-2-H,3-H]pyruvate, generated from 3(S)-]E-2-H,3-H]phosphoglycerate, was carboxylated and the oxalacetate trapped as [3030H]malate using malate dehydrogenase. Exhaustive incubation of the tritiated malate (3-H/14-C = 1.95) with fumarase to labilize the pro-R hydrogen at C-3 resulted in release of 65% of the tritium into water. Reisolation of the malate after fumarase action yielded a 30H/14-C ration of 0.67, indicating 34% retention as expected. The theoretical enantiotopic distribution for the observed k1H/k2H of 2.1 is 68:32. Selective enrichment of tritium in the pro-R position at C-3 of malate indicates enzymatic carboxylation of pyruvate with retention of configuration in this half-reaction also.     

s-Cis and s-trans isomerism of the His-Pro peptide bond in angiotensin and thyroliberin analogues.

The dipeptide His-Pro isomerizes from all-s-trans to partly s-cis when titrated in D2O from acidic to neutral pD as observed by 13C and 1H nuclear magnetic resonance of the proline side chain. This isomerization is reported by the His C-2 and C-4 protons and carbons which show distinct, well-resolved resonances for each isomer. The influence of the His-Pro peptide bond rotational state on the histidine protons far removed from the bond has not been previously observed in model compounds or peptides. The peptides thyroliberin (TRH), [3-MeHis2]-TRH, and [3-MeHis6]-, [Sar1,Al8]-, and Nalpha-acetylangiotensin II were found to similarly isomerize from all-s-trans to partly s-cis as reported by their His C-2 and C-4 proton resonances. The His C-2 and C-4 protons in the peptides [1,3-diMeHis2]-TRH and [1-MeHis6]-, and [homoHis6]-angiotensin do not report this isomerization. Angiotensin II has previously been found to exhibit the same isomerization. The reporting of the s-trans to s-cis isomerization by the His C-2 proton appears to be correlated with the known potencies of the five angiotensin peptides in rat uterine strips and of the three TRH peptides by radioimmunoassay of released thyrotropin.     

Stereochemistry and kinetic isotope effects in the bovine plasma amine oxidase catalyzed oxidation of dopamine.

The stereochemistry of the bovine plasma amine oxidase catalyzed oxidation of 2-(3,4-dihydroxyphenyl)-ethylamine (domapine) has been investigated by comparing 3H/14C ratios of 3,4-dibenzyloxyphenethyl alcohols, derived from 3,4-dihydroxyphenylacetaldehydes, to starting dopamines chirally labeled at C-1 and C-2. The oxidation of [2RS-3H]-, [2R-3H]-, and [2S-3H]dopamine leads to products which have retained 53, 59, and 47% of their tritium. Similarly, oxidation of [1RS-3H]-, [1R-3H]-, and [1S-3H]dopamine leads to an 80, 80, and 92% retention of tritium. The configurational purity of tritium at C-2 of dopamine and C-1 of the dopamine precursor 3-methoxy-4-hydroxyphenethylamine has been confirmed employing dopamine-beta-hydroxylase (specific for the pro-R hydrogen at C-2) and pea seedling amine oxidase (specific for the pro-S hydrogen at C-1). In addition, chromatographically resolved isozymes of bovine plasma amine oxidase have been demonstrated to lead to the same stereochemical result as pooled enzyme fractions. We have been able to rule out carbon interchange and tritium transfer in the ethylamine side chain of dopamine as the source of the apparent nonstereospecificity. Estimated primary tritium isotope effects are 1 for [2-3H]dopamines and 5--6 and 26--34 for [1R-3H]- and [1S-3H]dopamine, respectively. We propose the presence of alternate dopamine binding modes, characterized by absolute but opposing stereochemistries and differential primary tritium isotope effects at C-1.     

Cytotoxicity activity of L-proline analogues of anthraquinone-2-carboxylic acid in breast cancer MCF-7 cells

Although prolidase [E.C.3.4.13.9] is found in normal cells, substantially increased levels are found in some neoplastic tissues. Prolidase evokes the ability to hydrolyse the imido-bond of various low molecular weight compounds coupled to L-proline. The synthesis of three proline analogues of anthraquinone-2-carboxylic acid (1-3) has been performed. Treatment of these prodrugs with prolidase generated L-proline and the free drug, demonstrating their substrate susceptibility prolidase. The concentrations of 1, 2 and 3 needed to inhibit [1H]thymidine incorporation into DNA by 50% (IC50) in breast cancer MCF-7 cells were found to be 185 +/- 5 microM, 107 +/- 6 microM and 87 +/- 6 microM, respectively, suggesting a lower cytotoxic potency of these compounds compared to Hoechst 33228 (IC50 = 55 +/- 6 microM).     

Metabolism of D-glucose anomers in rat pancreatic islets exposed to equilibrated D-glucose.

This study aims at establishing the contribution of alpha- and beta-D-glucose to the total generation of (3)HOH by rat pancreatic islets exposed to D-[2 - (3)H]glucose or D-[5 - (3)H] glucose at anomeric equilibrium. The islets were incubated for 60 min at 4 degrees C in the presence of equilibrated D-glucose (2.8 and 8.3 mM) mixed with tracer amounts of either alpha- or beta-D-glucose labelled with tritium on either the C (2) or C (5) of the hexose. Relative to their respective concentrations, (3)HOH generation from the anomers labelled with tritium on the C (2) or C (5) of the hexose provided beta/alpha ratios comparable to those previously found at both 2.8 and 8.3 mM, when the islets were exposed to each anomer separately. The relative contributions of each anomer to the total generation of (3)HOH was also close to the theoretical values derived from mathematical models for the catabolism of D-glucose at anomeric equilibrium in rat islets at both 2.8 and 8.3 mM and in the case of both D-[2 - (3)H]glucose and D-[5 - (3)H]glucose. Thus, even in islets exposed to D-glucose at anomeric equilibrium, the metabolic fate of alpha-D-glucose differs vastly from that of beta-D-glucose, the enzyme-to-enzyme channelling between hexokinase isoenzymes, especially glucokinase, and phosphoglucoisomerase being restricted to alpha-D-glucose 6-phosphate.     

Use of 5-deazaFAD to study hydrogen transfer in the D-amino acid oxidase reaction.

The apoprotein of hog kidney D-amino acid oxidase was reconstituted with 5-deazaflavin adenine dinucleotide (5-deazaFAD) to yield a protein which contains 1.5 mol of 5-deazaFAD/mol of enzyme. The deazaFAD-containing enzyme forms complexes with benzoate, 2-amino benzoate, and 4-aminobenzoate which are both qualitatively and quantitatively similar to those observed with native enzyme. The complex with 2-aminobenzoate exhibits a new long wavelength absorption band characteristic of a flavin charge-transfer complex. The reconstituted enzyme exhibits no activity when assayed by D-alanine oxidation. However, the bound chromophore can be reduced by alanine, phenylalanine, proline, methionine, and valine, but not by glutamate or aspartate, indicating the deazaFAD enzyme retains the substrate specificity of the native enzyme. Reduction of the enzyme by D-alanine exhibits a 1.6-fold deuterium isotope effect. Reoxidation of the reduced enzyme occurred in the presence of pyruvate plus ammonia, but not with pyruvate alone or ammonia alone. beta-Phenylpyruvate and alpha-ketobutyrate, but not alpha-ketoglutarate could replace pyruvate. Reduced enzyme isolated following reaction with [alpha-3H]alanine was found to contain 0.5 mol of tritium/mol of deazaFADH2. After denaturation of the tritium-labeled enzyme, the radioactivity was identified as deazaFADH2. Reaction of the reduced tritium-labeled enzyme with pyruvate plus ammonia prior to denaturation yields [alpha-3H]alanine and unlabeled deazaFAD. These results suggest that reduction and reoxidation of enzyme-bound deazaFAD involves the stereo-specific transfer of alpha-hydrogen from substrate to deazaFAD.