Carbon 13 NMR study of nonenzymatic reactions of pyridoxal 5'-phosphate with selected amino acids and of related reactions.
Carbon 13 nuclear magnetic resonance spectroscopy has been used to monitor the nonenzymatic reactions of pyridoxal 5'-phosphate with glycine, alanine, valine, serine, and with several other model compounds. Isotopically enriched amino acids were employed so that low concentrations could be utilized while still allowing relatively rapid acquisition of spectral data. The results for alanine and serine are particularly noteworthy in that alanine is deaminated to pyruvate and pyruvate is aminated to alanine, but contrary to the enzymatic reactions of various serine dehydratases wherein serine is converted to pyruvate, the nonenzymatic reaction utilizing serine results in hydroxypruvate rather than pyruvate formation. In the reverse reaction, hydroxypyruvate is aminated to serine but very inefficiently relative to the amination of pyruvate to alanine. The experimental results have been formulated into a proposed reaction mechanism for deamination of amino acids by pyridoxal-P. (+info)
Pyridoxamine, an inhibitor of advanced glycation reactions, also inhibits advanced lipoxidation reactions. Mechanism of action of pyridoxamine.
Maillard or browning reactions lead to formation of advanced glycation end products (AGEs) on protein and contribute to the increase in chemical modification of proteins during aging and in diabetes. AGE inhibitors such as aminoguanidine and pyridoxamine (PM) have proven effective in animal model and clinical studies as inhibitors of AGE formation and development of diabetic complications. We report here that PM also inhibits the chemical modification of proteins during lipid peroxidation (lipoxidation) reactions in vitro, and we show that it traps reactive intermediates formed during lipid peroxidation. In reactions of arachidonate with the model protein RNase, PM prevented modification of lysine residues and formation of the advanced lipoxidation end products (ALEs) N(epsilon)-(carboxymethyl)lysine, N(epsilon)-(carboxyethyl)lysine, malondialdehyde-lysine, and 4-hydroxynonenal-lysine. PM also inhibited lysine modification and formation of ALEs during copper-catalyzed oxidation of low density lipoprotein. Hexanoic acid amide and nonanedioic acid monoamide derivatives of PM were identified as major products formed during oxidation of linoleic acid in the presence of PM. We propose a mechanism for formation of these products from the 9- and 13-oxo-decadienoic acid intermediates formed during peroxidation of linoleic acid. PM, as a potent inhibitor of both AGE and ALE formation, may prove useful for limiting the increased chemical modification of tissue proteins and associated pathology in aging and chronic diseases, including both diabetes and atherosclerosis. (+info)
Chlorination of pyridinium compounds. Possible role of hypochlorite, N-chloramines, and chlorine in the oxidation of pyridinoline cross-links of articular cartilage collagen type II during acute inflammation.
Reactive oxygen species produced by activated neutrophils and monocytes are thought to be involved in mediating the loss of collagen and other matrix proteins at sites of inflammation. To evaluate their potential to oxidize the pyridinoline (Pyd) cross-links found in collagen types I and II, we reacted hydrogen peroxide (H(2)O(2)), hypochlorous acid/hypochlorite (HOCl/OCl(-)), and singlet oxygen (O(2)((1)delta g)) with the Pyd substitutes, pyridoxamine dihydrochloride and vitamin B(6), which share the same chemical structure and spectral properties of Pyd cross-links. Neither H(2)O(2) (125-500 microm) nor O(2)((1)delta g) (10-25 microm) significantly changed the spectral properties of pyridoxamine or vitamin B(6). Reaction of HOCl/OCl(-) (12.5-50 microm) with pyridoxamine at pH 7.2 resulted in a concentration-dependent appearance of two new absorbance peaks and a decrease in fluorescence at 400 nm (excitation 325 nm). The new absorbance peaks correlated with the formation of an N-chloramine and the product of its subsequent reaction with pyridoxamine. In contrast, the extent to which HOCl reacted with vitamin B(6), which lacks a primary amine group, was variable at this pH. At lysosomal pH 5.5, Cl(2)/HOCl/OCl(-) reacted with both pyridoxamine and vitamin B(6). Four of the chlorinated products of this reaction were identified by gas chromatography-mass spectrometry and included 3-chloropyridinium, an aldehyde, and several chlorinated products with disrupted rings. To evaluate the effects of Cl(2)/HOCl/OCl(-) on Pyd cross-links in collagen, we exposed bone collagen type I and articular cartilage type II to HOCl. Treatment of either collagen type with HOCl at pH 5. 0 or 7.2 resulted in the oxidation of amine groups and, for collagen type II, the specific decrease in Pyd cross-link fluorescence, suggesting that during inflammation both oxidations may be used by neutrophils and monocytes to promote the loss of matrix integrity. (+info)
Stereochemistry of the transamination reaction catalyzed by aminodeoxychorismate lyase from Escherichia coli: close relationship between fold type and stereochemistry.
Aminodeoxychorismate lyase is a pyridoxal 5'-phosphate-dependent enzyme that converts 4-aminodeoxychorismate to pyruvate and p-aminobenzoate, a precursor of folic acid in bacteria. The enzyme exhibits significant sequence similarity to two aminotransferases, D-amino acid aminotransferase and branched-chain L-amino acid aminotransferase. In the present study, we have found that aminodeoxychorismate lyase catalyzes the transamination between D-alanine and pyridoxal phosphate to produce pyruvate and pyridoxamine phosphate. L-Alanine and other D- and L-amino acids tested were inert as substrates of transamination. The pro-R hydrogen of C4' of pyridoxamine phosphate was stereospecifically abstracted during the reverse half transamination from pyridoxamine phosphate to pyruvate. Aminodeoxychorismate lyase is identical to D-amino acid aminotransferase and branched-chain L-amino acid aminotransferase in the stereospecificity of the hydrogen abstraction, and differs from all other pyridoxal enzymes that catalyze pro-S hydrogen transfer. Aminodeoxychorismate lyase is the first example of a lyase that catalyzes pro-R-specific hydrogen abstraction. The result is consistent with recent X-ray crystallographic findings showing that the topological relationships between the cofactor and the catalytic residue for hydrogen abstraction are conserved among aminodeoxychorismate lyase, D-amino acid aminotransferase and branched-chain L-amino acid aminotransferase [Nakai, T., Mizutani, H., Miyahara, I., Hirotsu, K., Takeda, S., Jhee, K.-H., Yoshimura, T., and Esaki, N. (2000) J. Biochem. 128, 29-38]. (+info)
Studies on mammalian ribonucleotide reductase inhibition by pyridoxal phosphate and the dialdehyde derivatives of adenosine, adenosine 5'-monophosphate, and adenosine 5'-triphosphate.
Ribonucleotide reductase activity in a partially purified enzyme preparation from Ehrilich tumor cells was inhibited by the dialdehyde derivatives of adenosine, 5-adenylic acid, and adenosine 5-triphosphate (prepared by the periodate oxidation of adenosine 5-adenylic acid, and adenosine 5-triphosphate). The borohydride-reduced derivative of periodate-oxidized adenosine was not inhibitory to the ribonucleotide reductase activity, showing that the aldehyde moiety was important in the inhibitory interactions of these compounds. This suggested the formation of a Schiff base between the dialdehyde derivative and an amino group (presumably, the epsilon-amino group of lysine). Pyridoxal phosphate, which is known to inhibit enzymes that have lysyl residues in the catalytic or allosteric sites, was an inhibitor of ribonucleotide reductase. Pyridoxal, pyridoxamine phosphate, pyridoxamine, and pyridoxine were not inhibitors. Borohydride reduction of the enzyme in the presence of pyridoxal phosphate produced a protein fraction that had little reductase activity remaining. The inhibition by pyridoxal phosphate was not influenced by increasing the substrate concentration (cytidine 5-diphosphate or adenosine 5-diphosphate), but was diminished by increasing the ratio of allosteric effector to pyridoxal phosphate concentrations, suggesting an interaction of pyridoxal phosphate at the regulatory site of ribonucleotide reductase. The addition of adenosine 5-triphosphate to the pyridoxal phosphate-enzyme mixture, which was subsequently treated with borohydride, partially prevented the inhibition by pyridoxal phosphate. Heat treatment of the ribonucleotide reductase enzyme preparation in the presence of pyridoxal phosphate protected the enzyme against loss of cytidine 5-diphosphate and adenosine 5-diphosphate reductase activities. (+info)
Assessment of vitamin B-6 status in young women consuming a controlled diet containing four levels of vitamin B-6 provides an estimated average requirement and recommended dietary allowance.
The Recommended Dietary Allowance (RDA) of vitamin B-6 for young women was recently reduced from 1.6 to 1.3 mg/d based on an adequate plasma pyridoxal phosphate (PLP) concentration of 20 nmol/L. To assess vitamin B-6 requirements and suggest recommendations for intake, seven healthy young women consumed a controlled diet providing 1.2 g protein/kg body weight for a 7-d adjustment period (1.0 mg vitamin B-6/d) and three successive 14-d experimental periods (1.5, 2.1 and 2.7 mg/d, respectively). Direct and indirect vitamin B-6 status indicators were measured in plasma, erythrocytes and urine. Indicators most strongly correlated with vitamin B-6 intake [i.e., plasma and erythrocyte PLP, urinary 4-pyridoxic acid (4-PA) and total vitamin B-6] were regressed on vitamin B-6 intake and the dietary vitamin B-6 to protein ratio. Inverse prediction using adequate and baseline values estimated vitamin B-6 requirement. Adequate values were determined for plasma PLP and urinary 4-PA from baseline values of 60 previous subjects, using the statistical method suggested by Sauberlich. The current study suggests a vitamin B-6 Estimated Average Requirement (EAR) for young women of 1.1 mg/d or 0.016 mg/g protein, and a RDA of 1.5 mg/d or 0.020 mg/g protein. When results from this study are combined with data from four other recent studies, the combined data predict an EAR of 1.2 mg/d or 0.015 mg/g protein, and a RDA of 1.7 mg/d or 0.018 mg/g protein. This study suggests that the current vitamin B-6 RDA may not be adequate. (+info)
Separation and evaluation of the covalent and noncovalent interactions which contribute to the binding of pyridoxal 5'-phosphate to D-serine apodehydratase.
The equilibrium constant (KX) for the reaction D-serine dehydratase + pyridoxamine-P in equilibrium KX D-serine apodehydratase: pyridoxamine-P + pyridoxal-P was determined. At 25 degreees, pH 7.80, KX increases from 5.4 times 10-minus 5 to 21 times 10-minus 5 as T/2 is increased from 0.33 to 0.66. A value of 1.3 times 10-minus 4 M at 25 degrees, pH 7.80, T/2 0.33 for the equilibrium constant (KPMP) for dissociation of pyridoxamine-P from D-serine apodehydratase was determined from the ratio of the equilibrium constant for dissociation of pyridoxal-P from holoenzyme to KX. Pyridoxamine-P and the thiazolidine, formed from pyridoxal-P and cysteine, were found to have similar affinities for D-serine apodehydratase. Using the affinities of these derivatives as a measure of the noncovalent interactions between cofactor and protein, it was possible to estimate the contribution of the Schiff base linkage to the stability of the complex formed between pyridoxal-P and protein. The covalent Schiff base linkage in the holoenzyme was found to be no more stable than the Schiff base linkage formed between 6-aminocaproic acid and pyridoxal-P. The contribution of noncovalent interactions to the stability of the cofactor-protein complex was shown to be at least 20 to 40 times greater than the contribution of the covalent Schiff base linkage. (+info)
Studies on new intracellular proteases in various organs of rat. 1. Purification and comparison of their properties.
1. Specific proteases which inactivate the apo-proteins of many pyridoxal enzymes were found in skeletal muscle, liver and small intestine of rats. The protease from these three organs were purified and their properties were compared. 2. The purified proteases from liver and skeletal muscle appeared homogeneous on acrylamide gel electrophoresis. Two different proteases were separated from small intestine. A homogeneous, crystalline enzyme was obtained from the muscle layer while enzyme from the mucosa was partially purified. 3. They showed substrate specificity for pyridoxal enzymes. Their pH optima were in an alkaline region. They showed activity with the substrate of chymotrypsin, N-acetyl-L-tyrosine ethyl ester, but not with that of trypsin, p-toluenesulfonyl-L-arginine ethyl ester. They were inhibited by pyridoxal phosphate or pyridoxamine phosphate and seryl residues were involved in their active center. 4. The four enzymes differed in the following characters: (a) molecular weights; (b) patterns of elution from a CM-Sephadex column; (c) rates of inactivation of substrate enzymes; (d) rates of cleavage of N-acetyl-L-tyrosine ethyl ester; (e) reactivities with antiserum against the enzyme from the muscle layer of small intestine; (f) specific activities. 5. The amino acid composition and effect of chemical modifications of the crystalline enzyme from the muscle layer of small intestine were examined to elucidate its active sites and mode of action. Serine and histidine residues were found to be essential for protease activity. A tyrosine residue was also necessary for activity. Modifications of its sulfhydryl group, amino residues and carboxyl group had no effect on its activity. (+info)