Stereochemical aspects of cholinesterase catalysis

Proceeding from the sterochemical regularities of the nucleophilic substitution reaction at the carbonyl group and the assumption that the spatial structure of the active center of cholinesterases is complementary to the molecule of the ester substrates for these enzymes, some general features of the stereoselectivity phenomena in the reactions of acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) with organophosphorus inhibitors are discussed. For these enzymes the models of the active center are proposed in terms of different binding sites and the catalytic center. On the basis of this model, the stereochemical pecularities and the physicochemical background of the stereoselectivity effects on enzyme inhibition, reactivation, and “aging” reactions can be understood. Knowledge of the absolute configuration of several chiral organophosphorus inhibitors also makes it possible to determine the absolute spatial arrangement of the hydrophobic binding sites on the active surface of cholinesterases.     

Stereochemical action of mouse brain glutamate decarboxylase

4-[4-²H]Aminobutyrate was prepared by incubation in ²H₂O of glutamate with a partially purified glutamate decarboxylase from mouse brain. The 4R configuration was assigned to the compound on the basis of ¹H nmr analysis of the ω-camphanoylamide of its methyl ester in the presence of Eu(dpm)₃. Moreover 4-[4(S)4-³H,U-¹⁴C]aminobutyrate was shown to be formed from [2(S)2-³H,U-¹⁴C]glutamate by the same enzyme fraction. It is therefore demonstrated that glutamate decarboxylation catalyzed by this enzyme preparation occurs with retention of configuration.     

Functions of malonate decarboxylase subunits from Pseudomonas putida

Malonate decarboxylase from Pseudomonasputida is composed of five subunits, alpha, beta, gamma, delta, and epsilon. Two subunits, delta and epsilon, have been identified as an acyl-carrier protein (ACP) and malonyl-CoA:ACP transacylase, respectively. Functions of the other three subunits have not been identified, because recombinant subunits expressed in Escherichia coi formed inclusion bodies. To resolve this problem, we used a coexpression system with GroEL/ES from E. coli, and obtained active recombinant subunits. Enzymatic analysis of the purified recombinant subunits showed that the alpha subunit was an acetyl-S-ACP:malonate ACP transferase and that the betagamma-subunit complex was a malonyl-S-ACP decarboxylase.     

Stereochemical studies on phosphopantothenoylcysteine decarboxylase from Escherichia coli

Phosphopantothenoylcysteine decarboxylase catalyzes the decarboxylation of 4'-phosphopantothenoylcysteine (2) to form 4'-phosphopanthetheine (3), an intermediate in the biosynthesis of Coenzyme A. In this study we investigated the stereochemistry of this reaction. Our results show that the decarboxylation proceeds with retention of stereochemistry, and that the pro-R proton at C(beta) of the cysteine moiety of 2 is removed during a reversible oxidation of the thiol to a thioaldehyde intermediate.     

Stereochemical course of tyramine oxidation by semicarbazide-sensitive amine oxidase.

Two semicarbazide-sensitive amine oxidases (SSAO's) from bovine and porcine aortic tissue were partially purified and characterized, and the stereochemical course of amine oxidation was evaluated. The porcine and bovine SSAO's were membrane bound glycoproteins, with Km values for benzylamine of 8 and 16 microM, respectively. The reactivity of SSAO with semicarbazide and phenylhydrazine suggests that the cofactor is a carbonyl type molecule. The stereochemical course of the bovine and porcine aortic semicarbazide-sensitive amine oxidase reaction was investigated using chiral tyramines, deuterated at C-1 and C-2, and 1H-NMR spectroscopy to establish the loss or retention of deuterium in product p-hydroxyphenethyl alcohols. The preferred mode of tyramine oxidation was found to occur with the loss of pro-S proton at C-1, coupled with solvent exchange into C-2, a pattern which has not been observed for any copper amine oxidase examined to date. The solvent exchange reaction also occurred stereospecifically, with loss from and reprotonation to the pro-R position, suggesting that these two processes occur from the same face of the enamine double bond.     

Cloning and sequencing of genes encoding malonate decarboxylase in Acinetobacter calcoaceticus

Malonate decarboxylase from Acinetobacter calcoaceticus was isolated and characterized (Kim, Y.S., Byun, H.S., J. Biol. Chem. 269 (1994) 29636–29641), and its subunits were reanalyzed recently to be α, β, γ, and δ. The genes for the subunits, MdcA (548 a.a.), B (295 a.a.), C (238 a.a.), and D (102 a.a.), of the enzyme have been cloned by using oligonucleotide primers deduced from amino acid sequences of peptides isolated from the purified enzyme, and sequenced to be clustered in an operon in the order of A-D-B-C. The operon was found to encode more genes than mdcABCD. The Escherichia coli, transformed with the vector containing the insert mdcADBC and about 1.7 kb of an upstream region, expressed the four subunits of the enzyme but the proteins did not show enzyme activity. It indicates that, like the enzymes from Malonomonas rubra and Klebsiella pneumoniae, more genes are needed for the formation of the functional malonate decarboxylase.     

The biotin protein MadF of the malonate decarboxylase from Malonomonas rubra

The gene for the biotin protein MadF of the Na⁺-pumping malonate decarboxylase from Malonomonas rubra was expressed in Escherichia coli together with the gene for the biotin ligase birA. MadF was partially purified from cell lysates by ammonium sulfate precipitation. Almost pure biotin protein was obtained by subsequent gel chromatography. With recombinant MadF, malonate decarboxylase activity of M. rubra cell extracts previously inactivated by avidin was recovered. Thus, the biological activity of recombinant MadF was proven. Despite the coexpression of birA, MadF was poorly biotinylated. This effect was not caused by an insufficient cofactor supply due to elevated protein levels at constant biotin uptake rates. Attempts to improve the cofactor incorporation were made by site-directed mutagenesis, by coexpression of madK, and by N-terminal elongation of MadF. These measures improved the fraction of MadF containing biotin to maximally 5%. These results might indicate the existence of a biotin ligase in M. rubra with an altered substrate specificity different from that of BirA. Received: 4 June 1998 / Accepted: 7 September 1998     

Stereochemical course of two arene-cis-diol dehydrogenases specifically induced in Pseudomonas putida.

Catabolism of nonphenolic arenes is frequently initiated by dioxygenases, yielding single isomer products with two adjacent hydroxylated asymmetric centers. The next enzymic reaction dehydrogenates these cyclic cis-diols, with aromatization yielding catechols for ring cleavage. There are two stereochemical questions to answer. (i) To which face of NAD is hydride transferred giving NADH? (ii) Which hydrogen of the arene-cis-diols is donated to NAD? We report the results of 1H nuclear magnetic resonance [1H NMR] experiments for two diol dehydrogenases induced during growth of Pseudomonas putida PaW1(TOL) and JT105 with p-xylene and p-toluate, respectively. per-[2H5]benzoate-1,2-dihydrodiol and per-[2H7]- and specifically [2H]p-toluate-2,3-dihydrodiols were the substrates used to examine this by 1H NMR, as the two protons of the prochiral center (C-4 of the nicotinamide ring) are easily distinguished in the region of 2.6 to 2.7 ppm. We found that with the partially purified dehydrogenases (i) 2H from the (2R) center of per-(1S,2R)-benzoate-1,2-dihydrodiol was donated to the Si-face of NAD to give (4S)-NAD2H; (ii) p-toluate-2,3-diol dehydrogenase also provided exclusively (4S)-NAD2H, but the 2H was transferred from both the 2- and 3-C atoms of (2S,3R)-p-toluate-2,3-dihydrodiol with specifically deuterated species in approximately equal amounts; and (iii) the unexpected lack of stereo- and regioselectivity of p-toluate-2,3-diol dehydrogenase was supported by kinetic isotope effect studies.     

Oxalate decarboxylase uses electron hole hopping for catalysis

The hexameric low-pH stress response enzyme oxalate decarboxylase catalyzes the decarboxylation of the oxalate mono-anion in the soil bacterium Bacillus subtilis. A single protein subunit contains two Mn-binding cupin domains, and catalysis depends on Mn(III) at the N-terminal site. The present study suggests a mechanistic function for the C-terminal Mn as an electron hole donor for the N-terminal Mn. The resulting spatial separation of the radical intermediates directs the chemistry toward decarboxylation of the substrate. A π-stacked tryptophan pair (W96/W274) links two neighboring protein subunits together, thus reducing the Mn-to-Mn distance from 25.9 Å (intrasubunit) to 21.5 Å (intersubunit). Here, we used theoretical analysis of electron hole-hopping paths through redox-active sites in the enzyme combined with site-directed mutagenesis and X-ray crystallography to demonstrate that this tryptophan pair supports effective electron hole hopping between the C-terminal Mn of one subunit and the N-terminal Mn of the other subunit through two short hops of ∼8.5 Å. Replacement of W96, W274, or both with phenylalanine led to a large reduction in catalytic efficiency, whereas replacement with tyrosine led to recovery of most of this activity. W96F and W96Y mutants share the wildtype tertiary structure. Two additional hole-hopping networks were identified leading from the Mn ions to the protein surface, potentially protecting the enzyme from high Mn oxidation states during turnover. Our findings strongly suggest that multistep hole-hopping transport between the two Mn ions is required for enzymatic function, adding to the growing examples of proteins that employ aromatic residues as hopping stations.     

Inactivation of malonate semialdehyde decarboxylase by 3-halopropiolates: evidence for hydratase activity

Malonate semialdehyde decarboxylase (MSAD) from Pseudomonas pavonaceae 170 catalyzes the metal ion-independent decarboxylation of malonate semialdehyde and represents one of three known enzymatic activities in the tautomerase superfamily. The characterized members of this superfamily are structurally homologous proteins that share a beta-alpha-beta fold and a catalytic amino-terminal proline. Sequence analysis, chemical labeling studies, site-directed mutagenesis, and NMR studies of MSAD identified Pro-1 as a key active site residue in which the amino group has a pKa value of 9.2. The available evidence suggests a mechanism involving polarization of the C-3 carbonyl group of malonate semialdehyde by the cationic Pro-1. A second critical active site residue, Arg-75, could assist in the reaction by placing the substrate's carboxylate group in a favorable conformation for decarboxylation. In addition to the decarboxylase activity, MSAD has a hydratase activity as demonstrated by the MSAD-catalyzed conversion of 2-oxo-3-pentynoate to acetopyruvate. In view of this activity, MSAD was incubated with 3-bromo- and 3-chloropropiolate, and the subsequent reactions were characterized. Both compounds result in the irreversible inactivation of MSAD, making them the first identified inhibitors of MSAD. Inactivation by 3-chloropropiolate occurs in a time- and concentration-dependent manner and is due to the covalent modification of Pro-1. The proposed mechanism for inactivation involves the initial hydration of the 3-halopropiolate followed by a rearrangement to an alkylating agent, either an acyl halide or a ketene. The results provide additional evidence for the hydratase activity of MSAD and further support for the hypothesis that MSAD and trans-3-chloroacrylic acid dehalogenase, the preceding enzyme in the trans-1,3-dichloropropene catabolic pathway, diverged from a common ancestor but conserved the necessary catalytic machinery for the conjugate addition of water.     

Stereochemical course of DNA hydrolysis by nuclease S1

Nuclease S1 hydrolyzes the Sp-diastereomer of 5'-O-(2'-deoxyadenosyl)-3'-O-thymidyl phosphorothioate in H2(18)O to [18O]deoxyadenosine 5'-O-phosphorothioate which can be phosphorylated enzymatically to the Sp-diastereomer of [alpha-18O]deoxyadenosine 5'-O-(1-thiotriphosphate). 31P nmr spectroscopy shows the oxygen-18 in this compound to be in a nonbridging position at the alpha-phosphorus, indicating that the hydrolysis reaction catalyzed by nuclease S1 proceeds with inversion of configuration at phosphorus. This result is compatible with a direct nucleophilic attack of H2O at phosphorus without the involvement of a covalent enzyme intermediate.     

Biosynthesis of triphosphoribosyl-dephospho-coenzyme A, the precursor of the prosthetic group of malonate decarboxylase

Malonate decarboxylase from Klebsiella pneumoniae consists of four subunits MdcA, D, E, and C and catalyzes the cleavage of malonate to acetate and CO(2). The smallest subunit MdcC is an acyl carrier protein to which acetyl and malonyl thioester residues are bound via a 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group and turn over during the catalytic mechanism. We report here on the biosynthesis of holo acyl carrier protein from the unmodified apoprotein. The prosthetic group biosynthesis starts with the MdcB-catalyzed condensation of dephospho-CoA with ATP to 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA. In this reaction, a new alpha (1' ' --> 2') glycosidic bond between the two ribosyl moieties is formed, and thereby, the adenine moiety of ATP is displaced. MdcB therefore is an ATP:dephospho-CoA 5'-triphosphoribosyl transferase. The second protein involved in holo ACP synthesis is MdcG. This enzyme forms a strong complex with the 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA prosthetic group precursor. This complex, called MdcG(i), is readily separated from free MdcG by native polyacrylamide gel electrophoresis. Upon incubation of MdcG(i) with apo acyl carrier protein, holo acyl carrier protein is synthesized by forming the phosphodiester bond between the 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA prosthetic group and serine 25 of the protein. MdcG corresponds to a 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA:apo ACP 2'-(5' '-phosphoribosyl)-3'-dephospho-CoA transferase. In absence of the prosthetic group precursor, MdcG catalyzes at a low rate the adenylylation of apo acyl carrier protein using ATP as substrate. The adenylyl ACP thus formed is an unphysiological side product and is not involved in the biosynthesis of holo ACP. The 2'-(5' '-triphosphoribosyl)-3'-dephospho-CoA precursor of the prosthetic group has been purified and its identity confirmed by mass spectrometry and enzymatic analysis.     

Stereochemical course of the reaction catalyzed by 5'-nucleotide phosphodiesterase from snake venom.

The hydrolysis reaction of ATP alpha S by snake venom phosphodiesterase is highly specific for the B diastereomer and proceeds with 88% retention of configuration at phosphorus. Since this enzyme also catalyzes the hydrolysis of the S enantimoer of O-p-nitrophenyl phenylphosphonothioate, the absolute configuration at A alpha of ATP alpha S (B) is assigned as the R configuration provided the two substrates are processed identically. A mechanism for the hydrolysis reactions catalzyed by the venom phosphodiesterase involving at least a single covalent phosphoryl-enzyme intermediate is in accord with this result.     

Stereochemical course of thiophosphoryl transfer catalyzed by cytosolic phosphoenolpyruvate carboxykinase

Rat liver cytosolic phosphoenolpyruvate carboxykinase (PEPCK) utilizes inosine 5'-(3-thiotriphosphate) (ITP gamma S) as an excellent substrate, with Km and V values of 0.08 mM and 37 mumol min-1 (mg of protein)-1, respectively, compared with the corresponding values of 0.168 mM and 76 mumol min-1 (mg of protein)-1 for ITP. Thus, the V/Km values for the two substrates are the same. Reaction of (RP)-[gamma-18O2]ITP gamma S with oxalacetate catalyzed by cytosolic PEPCK produces (SP)-thio[18O]phosphoenolpyruvate. Therefore, thiophosphoryl transfer catalyzed by this enzyme proceeds with overall inversion of configuration at P. The reaction mechanism involves an uneven number of phosphotransfer steps, most likely a single step transfer between bound substrates. The results do not support the involvement of a phosphoryl enzyme intermediate in the mechanism.     

Myeloperoxidase inactivation in the course of catalysis of chlorination of taurine.

Myeloperoxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) was isolated from leukocytes of patients with chronic granulocyte leukemia. In the presence of H2O2 and Cl- at pH 4.0-6.6 the myeloperoxidase catalyses chlorination of taurine to monochloramine taurine and simultaneously undergoes inactivation. The myeloperoxidase inactivation rate depends on the concentration of H2O2 and Cl-: both the initial rate of chlorination and myeloperoxidase inactivation rate increase with increasing concentration of H2O2. However, an increase in concentration of Cl- results in a decrease in enzyme inactivation. At a given H2O2 concentration, myeloperoxidase inactivation is a first order reaction, which implied that the enzyme may react with a substrate a limited number of times.