Enzymatic in situ analysis by 1H-NMR of the hydrogen transfer stereospecificity of NAD(P)+-dependent dehydrogenases

We have established a simple procedure for the in situ analysis of stereospecificity of an NAD(P)-dependent dehydrogenase for C-4 hydrogen transfer of NAD(P)H by means of glutamate racemase [EC 5.1.13] and glutamate dehydrogenase [EC 1.4.1.3]. Glutamate racemase inherently catalyzes the exchange of alpha-H of glutamate with 2H during racemization in 2H2O. When the reactions of glutamate racemase and glutamate dehydrogenase, which is pro-S specific for the C4-H transfer of NAD(P)H, are coupled in 2H2O, [4S-2H]-NAD(P)H is exclusively produced. Therefore, if 1H is fully retained at C-4 of NAD(P)+ after incubation of a reaction mixture containing both the enzymes and a dehydrogenase to be tested, the stereospecificity of the dehydrogenase is the same as that of glutamate dehydrogenase. When the C4-H of NAD(P)+ is exchanged with 2H, the enzyme to be examined is different from glutamate dehydrogenase in stereospecificity. Thus, we can readily determine the stereospecificity by 1H-NMR measurement of NAD(P)+ without isolation of the coenzymes and products.     

Isotope effects in the study of enzymatic phosphoryl transfer reactions

CASK, a member of the membrane-associated guanylate kinase (MAGUK) superfamily, binds to the carboxyl-terminus of beta-neurexins on the intracellular side of the presynaptic membrane. The guanylate kinase-like (GUK) domains of MAGUKs lack kinase activities, but might be important for mediating specific protein-protein interaction. By a yeast two-hybrid approach, we identified an interaction between the GUK domain of CASK and the C2B domain of rabphilin3a, a presynaptic protein involved in synaptic vesicle exocytosis. The interaction was confirmed by in vitro GST pull-down and co-immunoprecipitation assays. It was proposed that presynaptic vesicles might be guided to the vicinity of points of exocytosis defined by beta-neurexins via the interaction between rabphilin3a-CASK-beta-neurexins.     

Enzymatic properties of human aminopeptidase A. Regulation of its enzymatic activity by calcium and angiotensin IV

Aminopeptidase A (APA) is a type II membrane-bound protein implicated in the regulation of blood pressure in the brain renin-angiotensin system. In this study, a recombinant soluble form of APA was expressed in a baculovirus system, purified to homogeneity, and characterized. By using synthetic substrates, it was shown that although the enzyme has a rather broad substrate specificity in the absence of Ca2+, the preferential release of acidic amino acid residues was observed in the presence of Ca2+. Moreover, Ca2+ up- or down-regulated the enzymatic activity depending on the substrate. By searching for natural substrates of APA, we found that peptides having acidic amino acids at their N terminus (angiotensin II, neurokinin B, cholecystokinin-8, and chromogranin A) were cleaved by the enzyme efficiently in the presence but not in the absence of Ca2+. Moreover kallidin (Lys-bradykinin) was converted to bradykinin effectively only in the absence of Ca2+. These results suggest that Ca2+ increases the preference of the enzyme for the peptide substrates having N-terminal acidic amino acids. In addition, we found that angiotensin IV could bind to APA both in the presence and absence of Ca2+ and inhibited the enzymatic activity of APA competitively, suggesting that angiotensin IV acts as a negative regulator of the enzyme once generated from angiotensin II by the serial actions of aminopeptidases. Taken together, these results suggest that there exists a complex regulation of the enzymatic activity of APA, which may contribute to homeostasis such as regulation of blood pressure, maintenance of memory, and normal pregnancy by controlling the concentrations of peptide substrates.     

Structural determination and stereospecificity of the choleragen-catalyzed reaction of NAD+ with guanidines.

The products of the choleragen-catalyzed reaction of NAD+ with guanidine HCl or L-arginine have been isolated by high performance liquid chromatography. Analysis of 1H NMR spectra obtained at 360 MHz establishes the structure of the isolated products to be adenosine diphosphoribosyl guanidiniums present as 1:1 mixtures of alpha and beta anomeric forms. Direct observation of the choleragen-catalyzed reaction of NAD+ with L-arginine by 1H NMR spectroscopy establishes that choleragen synthesizes an alpha anomeric linkage that is subject to subsequent anomerization. The stereospecificity of the reaction provides further evidence that choleragen behaves as an enzyme and may be related mechanistically to other ADP-ribosyltransferases.     

Intermolecular tritium transfer in the transcarboxylase reaction.

Transfer of tritium from [3-3H]pyruvate into propionyl-CoA is found during the reaction of transcarboxylase: Methylmalonyl-CoA + pyruvate leads to oxalacetate + propionyl-CoA. About 5% of the tritium counts that are labilized in the reaction are found in a position of the propionate that exchanges rapidly with water in the presence of transcarboxylase. Transfer from [2-3H]propionate of propionyl-CoA to pyruvate is real but only about one-tenth as great. The tritium transfers between reactants on two subunits are difficult to explain by a "carbanion" mechanism of --C--H bond cleavage and support the cyclic mechanism in which carboxybiotin itself is the base and the enol form of biotin is the proton-transferring agent.     

Determination of the absolute stereospecificity of the enzymatic reduction of NAD+ analogs by means of the primary acid cyclization reaction

NADH, (3-acetylpyridine)ADH, (3-thionicotinamide)ADH and (pyridine-3-aldehyde)ADH were labeled with deuterium by the B specific enzyme lipoyl dehydrogenase. The primary acid rearrangement products, α-O^2′-6B cyclotetra-hydropyridine adenine dinucleotides, of the respective reduced coenzyme analogs were synthesized by incubation at pH 2–2.5. Analysis of the spin-spin coupling constants of the 220 MHz proton magnetic resonance spectra establishes that the structure of the ribose-tetrahydropyridine linkage of these analogs is the same as that of the primary acid product of NADH. All four specifically labeled dinucleotides possess the identical absolute configuration at the C4 position with respect to the deuterium label, establishing that the three coenzyme analogs are reduced by lipoyl dehydrogenase with the same absolute stereospecificity as NAD⁺. The primary acid reaction is described as a general method for the determination of the absolute configuration at the C4 position of specifically labeled β-1,3-substituted, 1,4 dihydropyridine nucleotides.     

Enzymatic determination of galactosylceramide galactosidase in tissues by NAD cycling

An enzymatic determination method for galactosylceramide galactosidase (EC 3.2.1.46) was devised by using an enzymatic amplification reaction, NAD cycling. Galactose released by crude enzyme samples (tissue homogenates and cell suspensions) from galactosylceramide quantitatively reduced NAD to NADH by the galactose dehydrogenase reaction; then the NADH was amplified 6000-10,000-fold by NAD cycling and determined fluorometrically. A higher sensitivity of assay was obtained compared with the previous radiometric method. The present method was successfully applied to tissues from patients with Krabbe's disease, whose organs are deficient in galactosidase. The galactosidase reaction rate with a crude sample was not proportional to its concentration. However, the double-reciprocal plot of the reaction rate against the sample concentration became linear and provided a unique value of specific activity to each sample.     

Apparent noninvolvement of ADP-ribosyltransferases in nicotinamide production from NAD by porcine haemophili

Cell-free culture supernatant fractions derived from cultures ofHaemophilus parasuis andH. pleuropneumoniae failed to catabolize NAD irrespective of the presence or absence of dithiothreitol and/or arginine methyl ester. Although NAD was catabolized by resting cell suspensions of either organism and NMN, nicotinamide riboside, nicotinamide, adenosine, and adenine were detected as reaction products, the presence of dithiothreitol and/or arginine methyl ester in the reaction mixture made no obvious difference, qualitatively or quantitatively, to the results obtained. It is concluded that the production of nicotinamide from NAD by these organisms is due to the activities of conventional (i.e., nontoxigenic) enzymes rather than to toxins exhibiting ADP-ribosyltransferase activity.     

A stereochemical method for detection of ATP terminal phosphate transfer in enzymatic reactions. Glutamine synthetase.

An isotope scrambling method is described for the detection of transient [Enz:ADP:P-X] formation from [18O]ATP in ATP-coupled enzyme reactions. The method makes use of torsional symmetry of the newly formed (see article) group in ADP. [18 O]ATP labeled in the betagama bridge oxygen was incubated with enzyme and reversible cleavage of the PbetaO -- Pgamma bond was detected by the appearance of 18O in the beta nonbridge oxygens of the ATP pool. Experiments with sheep brain and Escherichia coli glutamine synthetases show that cleavage of ATP of enzyme-bound ADP and P-X requires glutamate. The exchange catalyzed by the E. coli enzyme with glutamate occurs in the absence of ammonia and is partially inhibited by added NH4Cl, as expected if the exchange is in the mechanistic pathway for glutamine synthesis. The results provide kinetic support for a two-step mechanism where phosphoryl transfer from ATP to glutamate precedes reaction with ammonia.     

Dimeric dihydrodiol dehydrogenase in monkey kidney. Substrate specificity, stereospecificity of hydrogen transfer, and distribution

Chemical cross-linking and NADPH binding studies suggested that the native dihydrodiol dehydrogenase from monkey kidney is a basic dimer having a molecular weight of 78,000 and one active site per the subunit. The enzyme oxidized specifically trans-dihydrodiols of benzene and naphthalene, whereas it catalyzed the reduction of dihydroxyacetone and dihydroxyacetone phosphate at a physiological pH, 7.4. The Km and kcat values for dihydroxyacetone phosphate were 5.0 mM and 4.3 s-1, respectively. The enzyme transferred the 4-pro-R hydrogen atom of NADPH to the carbonyl substrate. Immunochemical experiments using an antibody against the dimeric enzyme revealed the specific distribution of the enzyme in the kidney of this animal. By immunohistochemical staining with the specific antibody, the immunoreactivity was found in proximal and distal tubules of the cortex, and in the loop of Henle of the medulla.     

Determination of the hydride transfer stereospecificity of nicotinamide adenine dinucleotide linked oxidoreductases by proton magnetic resonance.

A facile proton magnetic resonance technique is described for the determination of the coenzyme stereospecificity during hydride transfer reactions catalyzed by pyridine nucleotide dependent oxidoreductases. The reliability of this technique was demonstrated by examining the coenzyme stereospecificity of lactate, malate, and 3-phosphoglycerate dehydrogenases, which are known to be A-stereospecific enzymes, as well as triosephosphate and octopine dehydrogenases, which are known to be B-stereospecific enzymes. Furthermore, by applying this technique, it was shown that the previously unstudied enzymes D-beta-hydroxybutyrate and 4-aminobutanal dehydrogenases are B- and A-stereospecific enzymes, respectively. In addition, the nicotinamide adenine dinucleotide linked reaction of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was found to be B stereospecific, like the reaction of the nicotinamide adenine dinucleotide phosphate linked yeast enzyme.     

Substrate specificity, regiospecificity and stereospecificity of halogenation reactions catalyzed by non-heme-type bromoperoxidase of Corallina pilulifera

Many organic compounds were found to be substrates for halogenation reactions catalyzed by the non-heme-type bromoperoxidase found in the red alga Corallina pilulifera. Anisole, 1-methoxynaphthalene and thiophene were converted to o and p-bromoanisoles, 1-methoxy-4-bromonaphthalene and 2-bromothiophene respectively. Regiospecificity of the enzymatic bromination of anisole was tested and found to be the same as in the chemical reaction with NaOBr. The enzyme also acted on substituted alkenes such as styrene, cyclohexene, trans-cinnamic acid, trans-cinnamyl alcohol and cis-propenylphosphonic acid, to give the respective bromohydrin compounds or decarboxylated bromo compound. These bromohydrin compounds were always mixtures of stereoisomers. In the light of the above findings together with the previous studies concerning the halogenation mechanism, the bromoperoxidase of C. pilulifera was considered to have no specific restriction site for these substrates.     

Determination of the accessible surface of globular proteins by means of tritium planigraphy

Results are presented for proteins with known three-dimensional structure (lysozyme, myoglobin, ribonuclease), which show that the probability of label incorporation upon bombardment by hot tritium atoms may be quantitatively linked with the surface area of the protein accessible to water molecules. Possible deviations from simple linear dependency caused by particular mechanisms of label introduction are discussed. The data obtained in experiments with model systems were used to determine the accessible surface area of human serum albumin, for which structural data is not sufficiently accurate to allow estimation of accessible surface area. Experimental data correlate reasonably well with estimations based on conventional concepts of the relationship between accessible surface area and molecular weight for globular proteins. Correspondence to: A. V. Volynskaya