Use of inhibitors to study reactions catalyzed by enzymes requiring pyridoxal phosphate as coenzyme

2000 ◽  
Vol 72 (3) ◽  
pp. 373-384 ◽  
Author(s):  
Benjamin Adams ◽  
B. Svante Axelsson ◽  
Kenneth J. M. Beresford ◽  
Nicola J. Church ◽  
Philip A. Spencer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Active Site ◽  
Aspartate Aminotransferase ◽  
Pyridoxal Phosphate ◽  
Family Formation ◽  
Enzyme Inhibitors ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Naturally Occurring

The stereochemistry of a variety of pyridoxal phosphate-mediated enzymic reactions has been studied using enzyme inhibitors that are stereospecifically labeled in the β-position with deuterium. A versatile synthesis has been developed to prepare a wide variety of stereospecifically labeled d- and l-amino acids and inhibitors. Investigation of the "turnover" of β-chloro-d-alanine and d- and l-serine-O-sulfate by d-amino acid aminotransferase and l-aspartate aminotransferase respectively has shown that reaction within the active site of the former enzyme occurs with retention of stereochemistry. Although l-aspartate aminotransferase is an enzyme of the α-family, when it was incubated with β-chloro-l-alanine in the presence of 2-mercaptoethanol, β-substitution occurred. This was shown to involve retention of stereochemistry, an outcome typical of reactions catalyzed by enzymes of the β-family that have little or no homology with enzymes of the α-family. Formation of the "Schnackerz intermediate" has been studied as has the d-amino acid oxidase catalyzed reaction of the naturally occurring inhibitor d-propargylglycine.

scholarly journals The mechanism of action of serine transhydroxymethylase

1970 ◽  
Vol 116 (2) ◽  
pp. 277-286 ◽  
Author(s):  
P. M. Jordan ◽  
M. Akhtar
Keyword(s):  
Schiff Base ◽  
Amino Acid ◽  
Hydrogen Atom ◽  
Mechanism Of Action ◽  
Active Site ◽  
Enzyme Preparation ◽  
Pyridoxal Phosphate ◽  
Acid Oxidase ◽  
Amino Acid Oxidase

1. The preparation of stereospecifically tritiated glycines and the determination of their absolute configurations by the use of d-amino acid oxidase are described. 2. The reaction catalysed by serine transhydroxymethylase, which results in the conversion of glycine into serine, has been separated into at least four partial reactions. It is suggested that the first event in this conversion is the formation of a Schiff base intermediate of glycine and pyridoxal phosphate. The next important step involves the removal of the 2S-hydrogen atom of glycine to give a carbanion intermediate. Experiments pertinent to the mechanism of conversion of this carbanion intermediate into serine are described. 3. The enzyme preparation catalysing the conversion of glycine into serine also participates in the conversion of glycine into threonine and allothreonine. In both these conversions, glycine → serine and glycine → threonine, the 2S-hydrogen atom of glycine is eliminated and the 2R-hydrogen atom of glycine is retained. 4. In the light of these experiments the mechanism of action of serine transhydroxymethylase is discussed. It is suggested that methylenetetrahydrofolate is the carrier of formaldehyde, from which formaldehyde may be liberated at the active site of the enzyme, thus allowing the overall reaction to take place.

scholarly journals Biological role of D-amino acid oxidase and D-aspartate oxidase. Effects of D-amino acids.

1993 ◽  
Vol 268 (36) ◽  
pp. 26941-26949
Author(s):  
A D'Aniello ◽  
G D'Onofrio ◽  
M Pischetola ◽  
G D'Aniello ◽  
A Vetere ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Biological Role ◽  
Acid Oxidase ◽  
Amino Acid Oxidase

scholarly journals Localization of D-amino acid oxidase on the cell surface of human polymorphonuclear leukocytes

1978 ◽  
Vol 77 (1) ◽  
pp. 59-71 ◽  
Author(s):  
JM Robinson ◽  
RT Briggs ◽  
MJ Karnovsky
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cell Surface ◽  
Polymorphonuclear Leukocytes ◽  
Ultrastructural Localization ◽  
H2o2 Production ◽  
Acid Oxidase ◽  
Resting Cells ◽  
Amino Acid Oxidase ◽  
Human Polymorphonuclear Leukocytes

The ultrastructural localization of D-amino acid oxidase (DAO) was studied cytochemically by detecting sites of hydrogen peroxide production in human polymorphonuclear leukocytes (PMNs). Reaction product, which forms when cerous ions react with H2O2 to form an electron-dense precipitate, was demonstrated on the cell surface and within the phagosomes of phagocytically stimulated cells when D-amino acids were provided as substrate. Resting cells showed only slight activity. The competitive inhibitor D,L-2-hydroxybutyrate greatly reduced the D-amino acid-stimulated reaction while KCN did not. The cell surface reaction was abolished by nonpenetrating inhibitors of enzyme activity while that within the phagosome was not eliminated. Dense accumulations of reaction product were formed in cells which phagocytosed Staphylococcus aureus in the absence of exogenous substrate. No reaction product formed with Proteus vulgaris while an intermediate amount formed when Escherichia coli were phagocytosed. Variation in the amount of reaction product with the different bacteria correlated with the levels of D-amino acids in the bacterial cell walls which are available for the DAO of PMNs. An alternative approach utilizing ferricyanide as an electron acceptor was also used. This technique verified the results obtained with the cerium reaction, i.e., the DAO is located in the cell surface and is internalized during phagocytosis and is capable of H2O2 production within the phagosome. The present finding that DAO is localized on the cell surface further supports the concept that the plasma membrane is involved in peroxide formation in PMNs.

Production ofD-amino acid oxidase byAspergillussp. strain O20 active on cephalosporin C

1997 ◽  
Vol 43 (3) ◽  
pp. 292-295 ◽  
Author(s):  
Salim K. Mujawar ◽  
Jaiprakash G. Shewale
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Stationary Growth Phase ◽  
Acid Oxidase ◽  
Cephalosporin C ◽  
Production Medium ◽  
Stationary Growth ◽  
Amino Acid Oxidase ◽  
Aspergillus Sp

Aspergillus sp. strain O20 produces inducible D-amino acid oxidase intracellularly, only in the presence of some amino acids. The enzyme was induced most effectively by the addition of DL-alanine (1% w/v) to the production medium. Among the various compounds studied, production of the D-amino acid oxidase was enhanced by Aerosol-22, glucose, and sodium nitrate. D-Amino acid oxidase formation was observed during the onset of the stationary growth phase. Maximum enzyme activity was recorded after 96 h of fermentation (1000 IU/L).Key words: D-amino acid oxidase, Aspergillus sp., 7-aminocephalosporanic acid, cephalosporin C.

An Automated Detection System for Most L-α-Amino Acids

1969 ◽  
Vol 15 (2) ◽  
pp. 154-161 ◽  
Author(s):  
K Van Dyke ◽  
C Szustkiewicz
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Detection System ◽  
Automated System ◽  
Acid Oxidase ◽  
Enzymatic Reactions ◽  
Amino Acid Oxidase ◽  
Automated Method ◽  
Wide Range

Abstract An automated system for the determination of the L-α form of the majority of amino acids is presented. The method is based upon oxidative deamination of the amino acid coupled with oxidation of o-dianisidine by hydrogen peroxide. This procedure can be used comparatively for the determination of a mixture of L-α-amino acids or for the majority of separated L-α-amino acids (especially in conjunction with column separations from urine and blood which give falsely positive identification with ninhydrin detection). The stereospecific nature of the L-α-amino acid oxidase enables the investigator to quantitate the amount of L-α-amino acid in the presence of the D-α form. From an academic viewpoint, the extreme sensitivity and wide range of the detection system make it advantageous for the study of the enzyme itself. This automated method also may be employed to follow enzymatic reactions—e.g., those catalyzed by peptidases or racemases. The methodology is extremely convenient with good reagent stability and is much more sensitive than manometric technics.

P219L substitution in human D-amino acid oxidase impacts the ligand binding and catalytic efficiency

2020 ◽  
Vol 168 (5) ◽  
pp. 557-567
Author(s):  
Wanitcha Rachadech ◽  
Yusuke Kato ◽  
Rabab M Abou El-Magd ◽  
Yuji Shishido ◽  
Soo Hyeon Kim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Conformational Changes ◽  
Active Site ◽  
Structural Changes ◽  
Catalytic Efficiency ◽  
Acid Oxidase ◽  
Wild Type ◽  
Amino Acid Oxidase ◽  
Adenine Ring ◽  
The Impact

Abstract Human D-amino acid oxidase (DAO) is a flavoenzyme that is implicated in neurodegenerative diseases. We investigated the impact of replacement of proline with leucine at Position 219 (P219L) in the active site lid of human DAO on the structural and enzymatic properties, because porcine DAO contains leucine at the corresponding position. The turnover numbers (kcat) of P219L were unchanged, but its Km values decreased compared with wild-type, leading to an increase in the catalytic efficiency (kcat/Km). Moreover, benzoate inhibits P219L with lower Ki value (0.7–0.9 µM) compared with wild-type (1.2–2.0 µM). Crystal structure of P219L in complex with flavin adenine dinucleotide (FAD) and benzoate at 2.25 Å resolution displayed conformational changes of the active site and lid. The distances between the H-bond-forming atoms of arginine 283 and benzoate and the relative position between the aromatic rings of tyrosine 224 and benzoate were changed in the P219L complex. Taken together, the P219L substitution leads to an increase in the catalytic efficiency and binding affinity for substrates/inhibitors due to these structural changes. Furthermore, an acetic acid was located near the adenine ring of FAD in the P219L complex. This study provides new insights into the structure–function relationship of human DAO.

Engineering the Substrate Specificity of Porcine Kidney d-Amino Acid Oxidase by Mutagenesis of the “Active-Site Lid”

2006 ◽  
Vol 139 (5) ◽  
pp. 873-879 ◽  
Author(s):  
Chiaki Setoyama ◽  
Yasuzo Nishina ◽  
Hisashi Mizutani ◽  
Ikuko Miyahara ◽  
Ken Hirotsu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Active Site ◽  
Acid Oxidase ◽  
Porcine Kidney ◽  
Amino Acid Oxidase
Sign in / Sign up

Export Citation Format

Share Document