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

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
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.

Identification of the active site and characterization of a novel sporulation-specific cysteine protease YabG from Bacillus subtilis

2021 ◽  
Author(s):  
Ryuji Yamazawa ◽  
Ritsuko Kuwana ◽  
Kenji Takeuchi ◽  
Hiromu Takamatsu ◽  
Yoshitaka Nakajima ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Chemical Modification ◽  
Cysteine Protease ◽  
Active Site ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Protease Gene ◽  
Active Site Residues ◽  
Protein Determination ◽  
35 Kda Protein

Abstract In order to characterize the probable protease gene yabG found in the genomes of spore-forming bacteria, Bacillus subtilis yabG was expressed as a 35 kDa His-tagged protein (BsYabG) in Escherichia coli cells. During purification using Ni-affinity chromatography, the 35 kDa protein was degraded via several intermediates to form a 24 kDa protein. Furthermore, it was degraded after an extended incubation period. The effect of protease inhibitors, including certain chemical modification reagents, on the conversion of the 35 kDa protein to the 24 kDa protein was investigated. Reagents reacting with sulfhydryl groups exerted significant effects, strongly suggesting that the yabG gene product is a cysteine protease with autolytic activity. Site-directed mutagenesis of the conserved Cys and His residues indicated that Cys218 and His172 are active site residues. No degradation was observed in the C218A/S and H172A mutants. In addition to the chemical modification reagents, benzamidine inhibited the degradation of the 24 kDa protein. Determination of the N-terminal amino acid sequences of the intermediates revealed trypsin-like specificity for YabG protease. Based on the relative positions of His172 and Cys218 and their surrounding sequences, we propose the classification of YabG as a new family of clan CD in the Merops peptidase database.

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.

Active-site mutations impairing the catalytic function of the catalytic subunit of human protein phosphatase 2A permit baculovirus-mediated overexpression in insect cells

2001 ◽  
Vol 357 (1) ◽  
pp. 225-232 ◽  
Author(s):  
Timothy MYLES ◽  
Karsten SCHMIDT ◽  
David R. H. EVANS ◽  
Peter CRON ◽  
Brian A. HEMMINGS
Keyword(s):  
Active Site ◽  
Protein Phosphatase ◽  
Insect Cells ◽  
Specific Activity ◽  
Catalytic Subunit ◽  
Regulatory Control ◽  
Wild Type ◽  
Active Site Residues ◽  
Catalytic Function

Members of the phosphoprotein phosphatase (PPP) family of protein serine/threonine phosphatases, including protein phosphatase (PP)1, PP2A and PP2B, share invariant active-site residues that are critical for catalytic function [Zhuo, Clemens, Stone and Dixon (1994) J. Biol. Chem. 269, 26234–26238]. Mutation of the active-site residues Asp88 or His118 within the human PP2A catalytic subunit (PP2Ac)α impaired catalytic activity in vitro; the D88N and H118N substitutions caused a 9- and 23-fold reduction in specific activity respectively, when compared with wild-type recombinant PP2Ac, indicating an important role for these residues in catalysis. Consistent with this, the D88N and H118N substituted forms failed to provide PP2A function in vivo, because, unlike wild-type human PP2Acα, neither substituted for the endogenous PP2Ac enzyme of budding yeast. Relative to wild-type PP2Ac, the active-site mutants were dramatically overexpressed in High Five® insect cells using the baculovirus system. Milligram quantities of PP2Ac were purified from 1×109 High Five cells and the kinetic constants for dephosphorylation of the peptide RRA(pT)VA (single-letter amino-acid notation) by PP2Ac (Km = 337.5μM; kcat = 170s−1) and D88N (Km = 58.4μM; kcat = 2s−1) were determined. The results show that the substitution impairs catalysis severely without a significant effect on substrate binding, consistent with the PPP catalytic mechanism. Combination of the baculovirus and yeast systems provides a strategy whereby the structure–function of PP2Ac may be fully explored, a goal which has previously proven difficult, owing to the stringent auto-regulatory control of PP2Ac protein levels in vivo.

scholarly journals Evidence that Asn542 of neprilysin (EC 3.4.24.11) is involved in binding of the P2′ residue of substrates and inhibitors

1995 ◽  
Vol 311 (2) ◽  
pp. 623-627 ◽  
Author(s):  
N Dion ◽  
H Le Moual ◽  
M C Fournié-Zaluski ◽  
B P Roques ◽  
P Crine ◽  
...  
Keyword(s):  
Active Site ◽  
Substrate Binding ◽  
Biologically Active ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
Hydrophobic Cluster Analysis ◽  
Active Peptides ◽  
Ic50 Values

Neprilysin (EC 3.4.24.11) is a Zn2+ metallopeptidase involved in the degradation of biologically active peptides, e.g. enkephalins and atrial natriuretic peptide. The substrate specificity and catalytic activity of neprilysin resemble those of thermolysin, a crystallized bacterial Zn2+ metalloprotease. Despite little overall homology between the primary structures of thermolysin and neprilysin, many of the amino acid residues involved in catalysis, as well as Zn2+ and substrate binding, are highly conserved. Most of the active-site residues of neprilysin have their homologues in thermolysin and have been characterized by site-directed mutagenesis. Furthermore, hydrophobic cluster analysis has revealed some other analogies between the neprilysin and thermolysin sequences [Benchetrit, Bissery, Mornon, Devault, Crine and Roques (1988) Biochemistry 27, 592-596]. According to this analysis the role of Asn542 in the neprilysin active site is analogous to that of Asn112 of thermolysin, which is to bind the substrate. Site-directed mutagenesis was used to change Asn542 to Gly or Gln residues. The effect of these mutations on substrate catalysis and inhibitor binding was examined with a series of thiorphan-like compounds containing various degrees of methylation at the P2′ residue. For both mutated enzymes, determination of kinetic parameters with [D-Ala2,Leu5]enkephalin as substrate showed that the large decrease in activity was attributable to an increase in Km (14-16-fold) whereas kcat values were only slightly affected (2-3-fold decrease). This is in agreement with Asn542 being involved in substrate binding rather than directly in catalysis. Finally, the IC50 values for thiorphan and substituted thiorphans strongly suggest that Asn542 of neprilysin binds the substrate on the amino side of the P2′ residue by formation of a unique hydrogen bond.

scholarly journals Heterologous expression of rat liver microsomal glutathione transferase in simian COS cells and Escherichia coli

1995 ◽  
Vol 311 (3) ◽  
pp. 861-866 ◽  
Author(s):  
R Weinander ◽  
E Mosialou ◽  
J DeJong ◽  
C P D Tu ◽  
J Dypbukt ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rat Liver ◽  
Expression Vector ◽  
Glutathione Transferase ◽  
Active Form ◽  
Coding Region ◽  
Western Blots ◽  
Cos Cells ◽  
Mammalian Expression ◽  
Microsomal Glutathione

The cDNA coding for rat liver microsomal glutathione transferase was subcloned into the mammalian expression vector pCMV-5 and the construct was transfected into, and transiently expressed in, simian COS cells. This resulted in high expression (0.7% of the microsomal protein). The activity towards 1-chloro-2,4-dinitrobenzene in microsomes was 15-30 nmol/min per mg, which increased upon N-ethylmaleimide treatment to 60-200 nmol/min per mg. Control and antisense-vector-treated cells displayed very low activity (3-6 nmol/min per mg). A DNA fragment coding for rat microsomal glutathione transferase was generated by PCR, cloned into the bacterial expression vector pSP19T7LT and transformed into Escherichia coli strain BL21 (DE3) (which contained the plasmid pLys SL). Isopropyl beta-D-thiogalactopyranoside (IPTG; 1 mM) induced the expression of significant amounts of enzymically active protein (4 mg/l of culture as measured by Western blots). The recombinant protein was purified and characterized and found to be indistinguishable from the rat liver enzyme with regard to enzymic activity, molecular mass and N-terminal amino acid sequence. Human liver cDNA was used to obtain the coding region of human microsomal glutathione transferase by PCR. This PCR product was cloned into pSP19T7LT, which, upon induction with IPTG, yielded significant amounts (9 mg/l of culture) of active enzyme in BL21 (DE3) cells. Thus, for the first time, it is now possible to express both human and rat microsomal glutathione transferase in an enzymically active form in Escherichia coli.

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