scholarly journals Identification of amino acid residues, essential for maintaining the tetrameric structure of sheep liver cytosolic serine hydroxymethyltransferase, by targeted mutagenesis

2003 ◽  
Vol 369 (3) ◽  
pp. 469-476 ◽  
Author(s):  
Venkatakrishna Rao JALA ◽  
Naropantul APPAJI RAO ◽  
Handanahal Subbarao SAVITHRI
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Serine Hydroxymethyltransferase ◽  
Targeted Mutagenesis ◽  
Amino Acid Residues ◽  
Oligomeric Structure ◽  
Subunit Interactions ◽  
Hydroxymethyl Group ◽  
Sheep Liver ◽  
Tetrameric Structure

Serine hydroxymethyltransferase (SHMT), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, catalyses the transfer of the hydroxymethyl group from serine to tetrahydrofolate to yield glycine and N5,N10-methylenetetrahydrofolate. An analysis of the known SHMT sequences indicated that several amino acid residues were conserved. In this paper, we report the identification of the amino acid residues essential for maintaining the oligomeric structure of sheep liver cytosolic recombinant SHMT (scSHMT) through intra- and inter-subunit interactions and by stabilizing the binding of PLP at the active site. The mutation of Lys-71, Arg-80 and Asp-89, the residues involved in intra-subunit ionic interactions, disturbed the oligomeric structure and caused a loss of catalytic activity. Mutation of Trp-110 to Phe was without effect, while its mutation to Ala resulted in the enzyme being present in the insoluble fraction. These results suggested that Trp-110 located in a cluster of hydrophobic residues was essential for proper folding of the enzyme. Arg-98 and His-304, residues involved in the inter-subunit interactions, were essential for maintaining the tetrameric structure. Mutation of Tyr-72, Asp-227 and His-356 at the active site which interact with PLP resulted in the loss of PLP, and hence loss of tetrameric structure. Mutation of Cys-203, located away from the active site, weakened PLP binding indirectly. The results demonstrate that in addition to residues involved in inter-subunit interactions, those involved in PLP binding and intra-subunit interactions also affect the oligomeric structure of scSHMT.

scholarly journals Identification of active-site residues of sheep liver serine hydroxymethyltransferase

1984 ◽  
Vol 224 (3) ◽  
pp. 703-707 ◽  
Author(s):  
R Manohar ◽  
N Appaji Rao
Keyword(s):  
Amino Acid ◽  
Chemical Modification ◽  
Active Site ◽  
Rate Constants ◽  
Second Order ◽  
Serine Hydroxymethyltransferase ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
Diethyl Pyrocarbonate ◽  
Sheep Liver

Chemical modification of amino acid residues with phenylglyoxal, N-ethylmaleimide and diethyl pyrocarbonate indicated that at least one residue each of arginine, cysteine and histidine were essential for the activity of sheep liver serine hydroxymethyltransferase. The second-order rate constants for inactivation were calculated to be 0.016 mM-1 X min-1 for phenylglyoxal, 0.52 mM-1 X min-1 for N-ethylmaleimide and 0.06 mM-1 X min-1 for diethyl pyrocarbonate. Different rates of modification of these residues in the presence and in the absence of substrates and the cofactor pyridoxal 5′-phosphate as well as the spectra of the modified protein suggested that these residues might occur at the active site of the enzyme.

scholarly journals Redox Properties of Human Medium-Chain Acyl-CoA Dehydrogenase, Modulation by Charged Active-Site Amino Acid Residues†

Biochemistry ◽  
1998 ◽  
Vol 37 (41) ◽  
pp. 14605-14612 ◽  
Author(s):  
Gina J. Mancini-Samuelson ◽  
Volker Kieweg ◽  
Kim Marie Sabaj ◽  
Sandro Ghisla ◽  
Marian T. Stankovich
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Redox Properties ◽  
Amino Acid Residues ◽  
Medium Chain ◽  
Chain Acyl

scholarly journals How [FeFe]-Hydrogenase Facilitates Bidirectional Proton Transfer

2019 ◽  
Author(s):  
Moritz Senger ◽  
Viktor Eichmann ◽  
Konstantin Laun ◽  
Jifu Duan ◽  
Florian Wittkamp ◽  
...  
Keyword(s):  
Amino Acid ◽  
Proton Transfer ◽  
Active Site ◽  
Molecular Hydrogen ◽  
H2 Production ◽  
Amino Acid Residues ◽  
Difference Spectroscopy ◽  
Dynamic Changes ◽  
In Situ Ir

Hydrogenases are metalloenzymes that catalyse the interconversion of protons and molecular hydrogen, H2. [FeFe]-hydrogenases show particularly high rates of hydrogen turnover and have inspired numerous compounds for biomimetic H2 production. Two decades of research on the active site cofactor of [FeFe]-hydrogenases have put forward multiple models of the catalytic proceedings. In comparison, understanding of the catalytic proton transfer is poor. We were able to identify the amino acid residues forming a proton transfer pathway between active site cofactor and bulk solvent; however, the exact mechanism of catalytic proton transfer remained inconclusive. Here, we employ in situ IR difference spectroscopy on the [FeFe]-hydrogenase from Chlamydomonas reinhardtii evaluating dynamic changes in the hydrogen-bonding network upon catalytic proton transfer. Our analysis allows for a direct, molecular unique assignment to individual amino acid residues. We found that transient protonation changes of arginine and glutamic acid residues facilitate bidirectional proton transfer in [FeFe]-hydrogenases.<br>

Roles of Active-Site Amino Acid Residues in Specific Recognition of DNA Lesions by Human 8-Oxoguanine-DNA Glycosylase (OGG1)

2019 ◽  
Vol 123 (23) ◽  
pp. 4878-4887 ◽  
Author(s):  
Timofey E. Tyugashev ◽  
Yury N. Vorobjev ◽  
Alexandra A. Kuznetsova ◽  
Maria V. Lukina ◽  
Nikita A. Kuznetsov ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Dna Lesions ◽  
Dna Glycosylase ◽  
Amino Acid Residues ◽  
Specific Recognition

scholarly journals Role of methionine in the active site of α-galactosidase from Trichoderma reesei

1995 ◽  
Vol 308 (3) ◽  
pp. 955-964 ◽  
Author(s):  
A M Kachurin ◽  
A M Golubev ◽  
M M Geisow ◽  
O S Veselkina ◽  
L S Isaeva-Ivanova ◽  
...  
Keyword(s):  
Amino Acid ◽  
Trichoderma Reesei ◽  
Active Site ◽  
Fold Increase ◽  
Thiol Group ◽  
Competitive Inhibitor ◽  
Amino Acid Residues ◽  
Activity Curve ◽  
Oxidative Activation ◽  
Alpha Galactosidase

alpha-Galactosidase from Trichoderma reesei when treated with H2O2 shows a 12-fold increase in activity towards p-nitrophenyl alpha-D-galactopyranoside. A similar effect is produced by the treatment of alpha-galactosidase with other non-specific oxidants: NaIO4, KMnO4 and K4S4O8. In addition to the increase in activity, the Michaelis constant rises from 0.2 to 1.4 mM, the temperature coefficient decreases by a factor of 1.5 and the pH-activity curve falls off sharply with increasing pH. Galactose (a competitive inhibitor of alpha-galactosidase; Ki 0.09 mM for the native enzyme at pH 4.4) effectively inhibits oxidative activation of the enzyme, because the observed activity changes are related to oxidation of the catalytically important methionine in the active site. NMR measurements and amino acid analysis show that oxidation to methionine sulphoxide of one of five methionines is sufficient to activate alpha-galactosidase. Binding of galactose prevents this. Oxidative activation does not lead to conversion of other H2O2-sensitive amino acid residues, such as histidine, tyrosine, tryptophan and cysteine. The catalytically important cysteine thiol group is quantitatively titrated after protein oxidative activation. Further oxidation of methionines (up to four of five residues) can be achieved by increasing the oxidation time and/or by prior denaturation of the protein. Obviously, a methionine located in the active site of alpha-galactosidase is more accessible. The oxidative-activation phenomenon can be explained by a conformational change in the active site as a result of conversion of non-polar methionine into polar methionine sulphoxide.

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