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 Identification of amino acid residues essential for enzyme activity of sheep liver 5,10-methylenetetrahydrofolate reductase

1986 ◽  
Vol 236 (1) ◽  
pp. 295-298 ◽  
Author(s):  
K Varalakshmi ◽  
H S Savithri ◽  
N A Rao
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Chemical Modification ◽  
Rate Constants ◽  
Methylenetetrahydrofolate Reductase ◽  
Second Order ◽  
Amino Acid Residues ◽  
Specific Chemical ◽  
Sheep Liver ◽  
Specific Chemical Modification

Sheep liver 5,10-methylenetetrahydrofolate reductase was subjected to specific chemical modification with phenylglyoxal, diethyl pyrocarbonate and N-bromosuccinimide. The second-order rate constants for inactivation were calculated to be 54 M-1 X min-1, 103 M-1 X min-1 and 154 M-1 X min-1 respectively. This inactivation could be prevented by incubation with substrates or products, suggesting that the residues modified, namely arginine, histidine and tryptophan, are essential for enzyme activity.

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

1990 ◽  
Vol 272 (2) ◽  
pp. 479-484 ◽  
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.

scholarly journals Comparative Characterization of CTX-M-64 and CTX-M-14 Provides Insights into the Structure and Catalytic Activity of the CTX-M Class of Enzymes

2016 ◽  
Vol 60 (10) ◽  
pp. 6084-6090 ◽  
Author(s):  
Dandan He ◽  
Jiachi Chiou ◽  
Zhenling Zeng ◽  
Edward Wai-Chi Chan ◽  
Jian-Hua Liu ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Amino Acid ◽  
Active Site ◽  
Structural Integrity ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
Significant Difference ◽  
Function Relationship ◽  
Alignment Analysis ◽  
M 14

ABSTRACTClinical isolates producing hybrid CTX-M β-lactamases, presumably due to recombination between theblaCTX-M-15andblaCTX-M-14elements, have emerged in recent years. Among the hybrid enzymes, CTX-M-64 and CTX-M-14 display the most significant difference in catalytic activity. This study aims to investigate the mechanisms underlying such differential enzymatic activities in order to provide insight into the structure/function relationship of this class of enzymes. Sequence alignment analysis showed that the major differences between the amino acid composition of CTX-M-64 and CTX-M-14 lie at both the N and C termini of the enzymes. Single or multiple amino acid substitutions introduced into CTX-M-64 and CTX-M-14 were found to produce only minor effects on hydrolytic functions; such a finding is consistent with the notion that the discrepancy between the functional activities of the two enzymes is not the result of only a few amino acid changes but is attributable to interactions between a unique set of amino acid residues in each enzyme. This theory is supported by the results of the thermal stability assay, which confirmed that CTX-M-64 is significantly more stable than CTX-M-14. Our data confirmed that, in addition to the important residues located in the active site, residues distal to the active site also contribute to the catalytic activity of the enzyme through stabilizing its structural integrity.

scholarly journals Identification of Active-Site Amino Acid Residues in the Chiba Virus 3C-Like Protease

2002 ◽  
Vol 76 (12) ◽  
pp. 5949-5958 ◽  
Author(s):  
Yuichi Someya ◽  
Naokazu Takeda ◽  
Tatsuo Miyamura
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Hepatitis A Virus ◽  
Side Chain ◽  
Significant Activity ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
3C Protease ◽  
Catalytic Dyad ◽  
Positively Charged

ABSTRACT The 3C-like protease of the Chiba virus, a Norwalk-like virus, is one of the chymotrypsin-like proteases. To identify active-site amino acid residues in this protease, 37 charged amino acid residues and a putative nucleophile, Cys139, within the GDCG sequence were individually replaced with Ala in the 3BC precursor, followed by expression in Escherichia coli, where the active 3C-like protease would cleave 3BC into 3B (VPg) and 3C (protease). Among 38 Ala mutants, 7 mutants (R8A, H30A, K88A, R89A, D138A, C139A, and H157A) completely or nearly completely lost the proteolytic activity. Cys139 was replaceable only with Ser, suggesting that an SH or OH group in the less bulky side chain was required for the side chain of the residue at position 139. His30, Arg89, and Asp138 could not be replaced with any other amino acids. Although Arg8 was also not replaceable for the 3B/3C cleavage and the 3C/3D cleavage, the N-terminal truncated mutant devoid of Arg8 significantly cleaved 3CD into 3C and 3D (polymerase), indicating that Arg8 itself was not directly involved in the proteolytic cleavage. As for position 88, a positively charged residue was required because the Arg mutant showed significant activity. As deduced by the X-ray structure of the hepatitis A virus 3C protease, Arg8, Lys88, and Arg89 are far away from the active site, and the side chain of Asp138 is directed away from the active site. Therefore, these are not catalytic residues. On the other hand, all of the mutants of His157 in the S1 specificity pocket tended to retain very slight activity, suggesting a decreased level of substrate recognition. These results, together with a sequence alignment with the picornavirus 3C proteases, indicate that His30 and Cys139 are active-site residues, forming a catalytic dyad without a carboxylate directly participating in the proteolysis.

scholarly journals Site-directed mutagenesis identified the key active site residues of alcohol acyltransferase PpAAT1 responsible for aroma biosynthesis in peach fruits

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Zhi-Zhong Song ◽  
Bin Peng ◽  
Zi-Xia Gu ◽  
Mei-Ling Tang ◽  
Bei Li ◽  
...  
Keyword(s):  
Amino Acid ◽  
Enzymatic Activity ◽  
Amino Acid Residue ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
Peach Fruit ◽  
Alcohol Acyltransferase

AbstractThe aroma of peach fruit is predominantly determined by the accumulation of γ-decalactone and ester compounds. A previous study showed that the biosynthesis of these aroma compounds in peach fruit is catalyzed by PpAAT1, an alcohol acyltransferase. In this work, we investigated the key active site residues responsible for γ-decalactone and ester biosynthesis. A total of 14 candidate amino acid residues possibly involved in internal esterification and 9 candidate amino acid residues possibly involved in esterification of PpAAT1 were assessed via site-directed mutagenesis. Analyses of the in vitro enzyme activities of PpAAT1 and its site-directed mutant proteins (PpAAT1-SMs) with different amino acid residue mutations as well as the contents of γ-decalactone in transgenic tobacco leaves and peach fruits transiently expressing PpAAT1 and PpAAT1-SMs revealed that site-directed mutation of H165 in the conserved HxxxD motif led to lost enzymatic activity of PpAAT1 in both internal esterification and its reactions, whereas mutation of the key amino acid residue D376 led to the total loss of γ-decalactone biosynthesis activity of PpAAT1. Mutations of 9 and 7 other amino acid residues also dramatically affected the enzymatic activity of PpAAT1 in the internal esterification and esterification reactions, respectively. Our findings provide a biochemical foundation for the mechanical biosynthesis of γ-decalactone and ester compounds catalyzed by PpAAT1 in peach fruits, which could be used to guide the molecular breeding of new peach species with more favorable aromas for consumers.

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