Chlorination of NADH: similarities of the HOCl-supported and chloroperoxidase-catalyzed reactions

The chloroperoxidase-catalyzed reactions of NAD(P)H with H2O2 in the presence of Cl- or Br- have been characterized. With 1 mol H2O2 per mol of NADH, one atom of 36Cl was incorporated into the 264-nm-absorbing intermediate product. This species was oxidized enzymatically by a second mole of H2O2 to a species distinct from NAD+, which retained one Cl atom. Spectroscopically identical species were also produced by reaction of NADH with one and two molar ratios of HOCl, respectively. These data indicate that, with respect to halogenation activities, chloroperoxidase functions similarly to myeloperoxidase, i.e., produces HOCl as the first product of Cl- oxidation by H2O2. Moreover, rapid chlorination of NAD(P)H followed by oxidation may be an important and highly lethal microbicidal effect of HOCl produced by myeloperoxidase in activated neutrophils.     

On the structure of flavin-oxygen intermediates involved in enzymatic reactions.

During the catalytic reactions of flavoprotein hydroxylases and bacterial luciferase, flavin peroxides are formed as intermediates [see Massey, V. and Hemmerich, P. (1976) in The Enzymes, 3rd edn (P. Boyer, ed.) pp. 421--505, Academic Press, New York]. These intermediates have been postulated to be C(4a) derivatives of the flavin coenzyme. To test this hypothesis, modified flavin coenzymes carrying an oxygen substituent at position C(4a) of the isoalloxazine ring were synthesized. They are tightly bound by the apoenzymes of D-amino acid oxidase, p-hydroxybenzoate hydroxylase and lactate oxidase; the resulting complexes show spectral properties closely similar to those of the transient oxygen adducts of the hydroxylases. The optical spectra of the lumiflavin model compounds were found to be highly dependent on the solvent environment and nature of the subsituents. Under appropriate conditions they simulate satisfactorily the spectra of the transient enzymatic oxygen adducts. The results support the proposal that the primary oxygen adducts formed with these flavoproteins on reaction of the reduced enzymes with oxygen are flavin C(4a) peroxides.     

Tunneling of intermediates in enzyme-catalyzed reactions

A fascinating group of enzymes has been shown to possess multiple active sites connected by intramolecular tunnels for the passage of reactive intermediates from the site of production to the site of utilization. In most of the examples studied to date, the binding of substrates at one active site enhances the formation of a reaction intermediate at an adjacent active site. The most common intermediate is ammonia, derived from the hydrolysis of glutamine, but molecular tunnels for the passage of indole, carbon monoxide, acetaldehyde and carbamate have also been identified. The architectural features of these molecular tunnels are quite different from one another, suggesting that they evolved independently.     

Thiiranium ion intermediates in the formation and reactions of S-(2-haloethyl)-l-cysteines

Thiiranium ions formed from the cysteine or glutathione conjugates of 1,2-dihaloethanes are believed to be responsible for the genotoxicity of the parent alkyl halides. The conversions of specifically deuterated β-hydroxyethyl sulfides to the corresponding β-haloethyl sulfides are studied to provide direct evidence for the involvement of thiiranium ions in the reactions of the cysteine conjugates of 1,2-dihaloethanes. S-(2-Hydroxyethyl-1, 1-d₂)-l-cysteine is converted to an equal mixture of the 1,1-d₂ and 2,2-d₂ isomers of the corresponding S-(2-haloethyl)-l-cysteines in concentrated hydrochloric, hydrobromic, or hydroiodic acids without detectable formation of the 2,2-d₂ isomer of the parent hydroxyethyl derivative. Dissolution of S-(2-hydroxyethyl)-l-cysteine in trifluoromethanesulfonic acid yields a compound with the NMR spectral properties of S-(l-cysteinyl)ethyl thiiranium trifluoromethanesulfonate. The organosoluble S-(2-hydroxyethyl-1,1-d₂) benzyl sulfide is converted to an equal mixture of the 1,1-d₂ and 2,2-d₂ isomers of S-(2-chloroethyl) benzyl sulfide by thionyl chloride or triphenylphosphine: carbon tetrachloride. These results demonstrate the involvement of thiiranium ion intermediates in the conversion of 2-hydroxyethyl sulfides to 2-haloethyl sulfides in halogen acids and a similar symmetrical intermediate in the chlorination reactions effected by thionyl chloride or triphenylphosphine: carbon tetrachloride.     

Immunogenetic analysis of H-2 mutations. III. Genetic mapping and involvement in immune reactions of the H-2ka mutation.

Mutation M523 (H-2ka) occurred spontaneously in strain CBA/CaLacSto and was discovered during routine skin graft testing for genetic homogeneity. By linkage and complementation tests, the mutation was previously mapped in the K end of the H-2 complex. We demonstrate that the mutation occurred in the K region, without affecting the I region in the K end of the complex. The mutant antigens cause rejection of skin grafts, stimulate cells in mixed lymphocyte culture, and function as stimulators as well as targets in cell-mediated lymphocytotoxicity. Yet, they are serologically indistinguishable from the antigens of the original strain and do not induce formation of humoral antibodies upon immunization of the CBA strain. Together with the results obtained on testing of other H-2 mutants, the data strongly support the notion that classical H-2 antigens (i.e., products of the H-2K and H-2D loci) can function as lymphocyte-stimulating determinants, and that I-region differences are not required for the induction of strong cell-mediated lymphocytotoxicity.     

Stabilization of noncovalent intermediates in enzymatically catalyzed reactions

Reactive intermediate enzyme complexes are difficult to study directly and the use of physical methods requiring observation periods of more than a second has not been possible heretofore. Here we introduce a simple approach, the "Le Chatelier forcing method" which does for the first time produce significant concentrations of such kinetically competent central intermediates observable for extended periods of time. The method involves only the forcing of the accumulation of intermediate complexes at thermodynamic equilibrium by the use of high reactant concentrations working against a high concentration of a product, combined with a valid and applicable method of analysis. We demonstrate this approach using the glutamate dehydrogenase catalyzed reaction with the reaction product ammonia as a "dam" to oppose the forward driving force of NADP and l-glutamate. We demonstrate the accumulation of substantial amounts measurable amounts of stable enzyme-NADPH-alpha-carbinolamine and alpha-iminoglutarate complexes in three different alpha-amino acid dehydrogenases. We describe the manipulation of such Le Chatelier forced equilibria to increase the prominence of particular species and discuss the implications of these findings for previously unattainable experimental approaches.     

Acyl intermediates in penicillopepsin-catalysed reactions and a discussion of the mechanism of action of pepsins.

Penicillopepsin catalyses transpeptidation reactions involving the transfer of the N-terminal amino acids of suitable substrates via covalent acyl intermediates to acceptor peptides, usually the substrate. The major products obtained when Phe-Tyr-Thr-Pro-Lys-Ala and Met-Leu-Gly were used as substrates were Phe-Phe and Met-Met respectively. With Met-Leu-Gly the tetrapeptide Met-Met-Leu-Gly was observed as probable intermediate. Co-incubation of Leu-Tyr-Leu and Phe-Tyr-Thr-Pro-Lys-Ala led to the formation of Leu-Phe and Phe-Leu as well as Leu-Leu and Phe-Phe. No reaction was observed with tripeptides in which the first or second amino acid is glycine. It appears that two amino aicds with large hydrophobic residues are needed for the transpeptidation reaction. Nucleophilic compounds other than peptides, such as hydroxylamine, aliphatic alcohols and dinitrophenylhydrazine, were not acceptors for the acyl group. Leucine, phenylalanine and leucine methyl ester also had no effect on the reaction. The transpeptidation reaction proceeded readily at pH 3.6 and 4.7. At pH 6.0 the reaction was slow and at pH 1.9 little or no transpeptidation was observed. Porcine pepsin catalyses similar transpeptidation reactions. Sequence studies show that porcine pepsin and penicillopepsin are homologous. The present study also suggests that they have a very similar mechanism. Evidence available at this time indicates that the mechanism of these enzymes is complex and may be modulated by secondary substrate-enzyme interactions. A hypothesis is presented which proposes that pepsin-catalysed reactions proceed via different covalent intermediates (amino-intermediates or acylintermediates) depending on the nature of the substrate. The possibility that some reactions do not involve covalent intermediates is also discussed.     

Acyl and amino intermediates in reactions catalysed by pig pepsin. Analysis of transpeptidation products.

The action of pig pepsin on a variety of small peptides including Leu-Trp-Met-Arg, Leu-Trp-Met, Leu-Leu-NH2, benzyloxycarbonyl-Phe-Leu and Gly-Leu-Tyr was studied. Leu-Leu-Leu was found to be the major product from the substrates Leu-Trp-Met-Arg and Leu-Trp-Met, indicating that the predominant reaction at pH 3.4 was a transpeptidation of the acyl-transfer type. Leu-Leu-Leu was also formed in high yield by amino transfer from benzyloxycarbonyl-Phe-Leu. Like the amino-transfer reactions the acyl transfer proceeded via a covalent intermediate, since [14C]leucine was not incorporated into transpeptidation products and did not exchange with enzyme-bound leucine in the presence of acceptors. With Leu-Trp-Met both acyl and amino transpeptidation products, namely Leu-Leu, Leu-Leu-Leu, Met-Met and Met-Met-Met, were formed in addition to methionine and leucine. With Leu-Trp-Met-Arg (1 mM) the pH optimum for the rates of hydrolysis and acyl transfer is about pH 3.4. At this pH the rate of acyl transfer exceeds that of hydrolysis; at pH 2, however, hydrolysis was faster than transfer. A comparison of the effect of the length of substrates and products on the reaction rates allows the conclusion that the binding site can extend over eight to nine amino acid residues. Although the experiments provide no conclusive evidence for or against the involvement of amino and/or acyl intermediates in the hydrolysis of long peptides and proteins, the high yield of transpeptidation reactions of both types observed with some substrates suggests a major role for the intermediates in pepsin-catalysed reactions. The results also show that when pig pepsin is used for the digestion of proteins for sequence work, the likelihood of the formation of transpeptidation products is considerable. In this way peptides not present in the original sequence could easily form in a reasonably good yield.     

A mass spectrometry-based probe of equilibrium intermediates in protein-folding reactions

Described here is a mass spectrometry- and H/D exchange-based approach for the detection of equilibrium intermediate state(s) in protein-folding reactions. The approach utilizes the stability of unpurified proteins from rates of H/D exchange (SUPREX) technique to measure the m value (i.e., delta DeltaG/delta [denaturant] value) associated with the folding reaction of a protein. Such SUPREX m-value analyses can be made over a wide range of denaturant concentrations. Thus, the described approach is well-suited for the detection of high-energy intermediates that might be populated at low denaturant concentrations and hard to detect in conventional chemical denaturation experiments using spectroscopic probes. The approach is demonstrated on four known non-two-state folding proteins, including alpha-lactalbumin, cytochrome c, intestinal fatty acid binding protein (IFABP), and myoglobin. The non-two-state folding behavior of each model protein system was detected by the described method. The cytochrome c, myoglobin, and IFABP systems each had high-energy intermediate states that were undetected in conventional optical spectroscopy-based studies and previously required other more specialized biophysical approaches (e.g., nuclear magnetic resonance spectroscopy-based methods and protease protection assays) for their detection. The SUPREX-based approach outlined here offers an attractive alternative to these other approaches, because it has the advantage of speed and the ability to analyze both purified and unpurified protein samples in either concentrated or dilute solution.     

Versatile syntheses of mono- or bis-allylhalothiol-carbonates of some alditol or sugar derivatives via cyclic thionocarbonates as intermediates

We describe herein an extension of the halogenation of 1,2 or 1,3-diols via a cyclic thionocarbonate functionality by reaction with an allyl halide instead of methyl iodide, which is usually used. This investigation was successfully carried out under both conventional heating and microwave solvent-free conditions with some alditol, thioanhydroalditol, and aldose derivatives.     

The involvement of carnitine intermediates in peroxisomal fatty acid oxidation: a study with 2-bromofatty acids

Metabolism-dependent inactivators of 3-ketothiolase I and carnitine acyltransferase I (CAT I) have been used to study the oxidation of fatty acids in intact hepatocytes. 2-Bromooctanoate inactivates mitochondrial and peroxisomal 3-ketothiolases I in a time-dependent manner. During the first 5 min of incubation, inactivation of 3-ketothiolase in mitochondria is five times faster than its inactivation in peroxisomes. Almost complete inactivation of 3-ketothiolase I in both types of organelle is achieved after incubation with 1 mM 2-bromooctanoate for 40 min. The inactivation is not affected by preincubating hepatocytes with 20 microM tetradecylglycidate (TDGA), an inactivator of CAT I, under conditions which cause greater than 95% inactivation of CAT I. 2-Bromododecanoate (1 mM) causes 60% inactivation of mitochondrial and peroxisomal 3-ketothiolases I in 40 min. These inactivations are greatly reduced by preincubating hepatocytes with 20 microM TDGA, demonstrating that 2-bromododecanoate enters both mitochondria and peroxisomes via its carnitine ester. 2-Bromopalmitate (1 mM) causes less than 5% inactivation of mitochondrial and peroxisomal 3-ketothiolases I in 40 min, but causes 95% inactivation of CAT I during this time. Incubation of hepatocytes with 10-200 microM 2-bromopalmitoyl-L-carnitine causes inactivation of mitochondrial and peroxisomal 3-ketothiolases I at similar rates. This inactivation is decreased by palmitoyl-D-carnitine during the first 5 min of incubation. Pretreating hepatocytes with 20 microM TDGA does not affect the inactivation of mitochondrial or peroxisomal 3-ketothiolase I by 2-bromopalmitoyl-L-carnitine. These results demonstrate that in intact hepatocytes, peroxisomes oxidize fatty acids of medium-chain length by a carnitine-independent mechanism, whereas they oxidize long-chain fatty acids by a carnitine-dependent mechanism.