Active-site Residues Governing High Steroid Isomerase Activity in Human Glutathione Transferase A3-3

hGSTZ1-1 (human glutathione transferase Zeta 1-1) catalyses a range of glutathione-dependent reactions and plays an important role in the metabolism of tyrosine via its maleylacetoacetate isomerase activity. The crystal structure and sequence alignment of hGSTZ1 with other GSTs (glutathione transferases) focused attention on three highly conserved residues (Ser-14, Ser-15, Cys-16) as candidates for an important role in catalysis. Progress in the investigation of these residues has been limited by the absence of a convenient assay for kinetic analysis. In this study we have developed a new spectrophotometric assay with a novel substrate [(±)-2-bromo-3-(4-nitrophenyl)propionic acid]. The assay has been used to rapidly assess the potential catalytic role of several residues in the active site. Despite its less favourable orientation in the crystal structure, Ser-14 was the only residue found to be essential for catalysis. It is proposed that a conformational change may favourably reposition the hydroxyl of Ser-14 during the catalytic cycle. The Cys16→Ala (Cys-16 mutated to Ala) mutation caused a dramatic increase in the Km for glutathione, indicating that Cys-16 plays an important role in the binding and orientation of glutathione in the active site. Previous structural studies implicated Arg-175 in the orientation of α-halo acid substrates in the active site of hGSTZ1-1. Mutation of Arg-175 to Lys or Ala resulted in a significant lowering of the kcat in the Ala-175 variant. This result is consistent with the proposal that the charged side chain of Arg-175 forms a salt bridge with the carboxylate of the α-halo acid substrates.

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Chemical modification of rat liver microsomal glutathione transferase defines residues of importance for catalytic function

Biochemical Journal ◽

10.1042/bj2720479 ◽

1990 ◽

Vol 272 (2) ◽

pp. 479-484 ◽

Cited By ~ 17

Author(s):

C Andersson ◽

R Morgenstern

Keyword(s):

Chemical Modification ◽

Rat Liver ◽

Active Site ◽

Glutathione Transferase ◽

Amino Acid Residues ◽

Active Site Residues ◽

Histidine Residues ◽

Catalytic Function ◽

Low Efficiency ◽

Microsomal Glutathione

Amino acid residues that are essential for the activity of rat liver microsomal glutathione transferase have been identified using chemical modification with various group-selective reagents. The enzyme reconstituted into phosphatidylcholine liposomes does not require stabilization with glutathione for activity (in contrast with the purified enzyme in detergent) and can thus be used for modification of active-site residues. Protection by the product analogue and inhibitor S-hexylglutathione was used as a criterion for specificity. It was shown that the histidine-selective reagent diethylpyrocarbonate inactivated the enzyme and that S-hexylglutathione partially protected against this inactivation. All three histidine residues in microsomal glutathione transferase could be modified, albeit at different rates. Inactivation of 90% of enzyme activity was achieved within the time period required for modification of the most reactive histidine, indicating the functional importance of this residue in catalysis. The arginine-selective reagents phenylglyoxal and 2,3-butanedione inhibited the enzyme, but the latter with very low efficiency; therefore no definitive assignment of arginine as essential for the activity of microsomal glutathione transferase can be made. The amino-group-selective reagents 2,4,6-trinitrobenzenesulphonate and pyridoxal 5′-phosphate inactivated the enzyme. Thus histidine residues and amino groups are suggested to be present in the active site of the microsomal glutathione transferase.

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Cys-X Scanning for Expansion of Active-site Residues and Modulation of Catalytic Functions in a Glutathione Transferase

Journal of Biological Chemistry ◽

10.1074/jbc.m111.230078 ◽

2011 ◽

Vol 286 (19) ◽

pp. 16871-16878 ◽

Cited By ~ 5

Author(s):

Malena A. Norrgård ◽

Ulf Hellman ◽

Bengt Mannervik

Keyword(s):

Active Site ◽

Glutathione Transferase ◽

Active Site Residues ◽

Catalytic Functions

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Faculty Opinions recommendation of 13C NMR analysis of electrostatic interactions between NAD+ and active site residues of UDP-galactose 4-epimerase: implications for the activation induced by uridine nucleotides.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1000833.9459 ◽

2001 ◽

Author(s):

Hung-Wen Liu

Keyword(s):

13C Nmr ◽

Active Site ◽

Electrostatic Interactions ◽

Nmr Analysis ◽

Active Site Residues ◽

13C Nmr Analysis ◽

Uridine Nucleotides

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Development of Xanthine Based Inhibitors Targeting Phosphodiesterase 9A

Letters in Drug Design & Discovery ◽

10.2174/1570180813666161102125423 ◽

2017 ◽

Vol 14 (10) ◽

pp. 1122-1137 ◽

Cited By ~ 1

Author(s):

Nivedita Singh ◽

Parameswaran Saravanan ◽

M.S. Thakur ◽

Sanjukta Patra

Keyword(s):

Free Energy ◽

Active Site ◽

Signal Transduction Pathway ◽

Docking Study ◽

Primary Objective ◽

Cgmp Level ◽

Active Site Residues ◽

Aromatic Fragment ◽

Docking Approach ◽

Free Energy Of Binding

Background: Phosphodiesterases 9A (PDE9A) is one of the prominent regulating enzymes of the signal transduction pathway having highest catalytic affinity for second messenger, cGMP. When the cGMP level is lowered, an uncontrolled expression of PDE9A may lead to various neurodegenerative diseases. To regulate the catalytic activity of PDE9A, potent inhibitors are needed. Objective: The primary objective of the present study was to develop new xanthine based inhibitors targeting PDE9A. This study was an attempt to bring structural diversification in PDE9A inhibitor development because most of the existing inhibitors are constructed over pyrazolopyrimidinone scaffold. Methods: Manual designing and parallel molecular docking approach were used for the development of xanthine derivatives. In this study, N1, N3, N9 and C8 positions of xanthine scaffold were selected as substitution sites to design 200 new compounds. Reverse docking and pharmaceutical analyses were used for final validation of most promising compounds. Results: By keeping free energy of binding cut-off of -6.0 kcal/mol, 52 compounds were screened. The compounds with substitution at N1, N3 and C8 positions of xanthine showed good occupancy in PDE9A active site pocket with a significant interaction pattern. This was further validated by screening different factors such as free energy of binding, inhibition constant and interacting active site residues in the 5Å region. Substitution at C8 position with phenyl substituent determined the inhibition affinity of compounds towards PDE9A by establishing a strong hydrophobic - hydrophobic interaction. The alkyl chain at N1 position generated selectivity of compounds towards PDE9A. The aromatic fragment at N3 position increased the binding affinity of compounds. Thus, by comparative docking study, it was found that compound 39-42 formed selective interaction towards PDE9A over other members of the PDE superfamily. Conclusion: From the present study, N1, N3 and C8 positions of xanthine were concluded as the best sites for substitution for the generation of potent PDE9A inhibitors.

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An enzymatic activation of formaldehyde for nucleotide methylation

Nature Communications ◽

10.1038/s41467-021-24756-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Charles Bou-Nader ◽

Frederick W. Stull ◽

Ludovic Pecqueur ◽

Philippe Simon ◽

Vincent Guérineau ◽

...

Keyword(s):

Amino Acids ◽

Thymidylate Synthase ◽

Active Site ◽

De Novo ◽

Crystallographic Structure ◽

De Novo Synthesis ◽

Active Site Residues ◽

Enzymatic Activation ◽

Enzyme Cofactors ◽

Unique Ability

AbstractFolate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.

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Covalent structure, disulfide bonding, and identification of reactive surface and active site residues of human prostatic acid phosphatase

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)52245-6 ◽

1991 ◽

Vol 266 (4) ◽

pp. 2313-2319

Author(s):

R L Van Etten ◽

R Davidson ◽

P E Stevis ◽

H MacArthur ◽

D L Moore

Keyword(s):

Acid Phosphatase ◽

Active Site ◽

Prostatic Acid Phosphatase ◽

Active Site Residues ◽

Disulfide Bonding ◽

Reactive Surface ◽

Covalent Structure

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Identification of two active site residues in human angiotensin I-converting enzyme.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)43897-5 ◽

1994 ◽

Vol 269 (47) ◽

pp. 29430-29434

Author(s):

T A Williams ◽

P Corvol ◽

F Soubrier

Keyword(s):

Active Site ◽

Converting Enzyme ◽

Active Site Residues ◽

Angiotensin I ◽

Angiotensin I Converting Enzyme

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Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/156.1.21 ◽

2000 ◽

Vol 156 (1) ◽

pp. 21-29 ◽

Cited By ~ 1

Author(s):

David R H Evans ◽

Brian A Hemmings

Keyword(s):

Protein Interactions ◽

Active Site ◽

Protein Function ◽

Mutational Analysis ◽

Yeast Cells ◽

Temperature Sensitive ◽

Active Site Residues ◽

Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

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