scholarly journals Enzymatic Methyl Transfer: Role of an Active Site Residue in Generating Active Site Compaction That Correlates with Catalytic Efficiency

2011 ◽  
Vol 133 (43) ◽  
pp. 17134-17137 ◽  
Author(s):  
Jianyu Zhang ◽  
Judith P. Klinman
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Active Site Residue ◽  
Methyl Transfer

The key role of a non-active-site residue Met148 on the catalytic efficiency of meta-cleavage product hydrolase BphD

2013 ◽  
Vol 97 (24) ◽  
pp. 10399-10411 ◽  
Author(s):  
Hao Zhou ◽  
Yuanyuan Qu ◽  
Chunlei Kong ◽  
E. Shen ◽  
Jingwei Wang ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Cleavage Product ◽  
Active Site Residue

scholarly journals Role of Non-Active-Site Residue Trp-93 in the Function and Stability of New Delhi Metallo-β-Lactamase 1

2015 ◽  
Vol 60 (1) ◽  
pp. 356-360 ◽  
Author(s):  
Asad U. Khan ◽  
M. Tabish Rehman
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Structure And Function ◽  
Clinical Strain ◽  
New Delhi ◽  
Active Site Residue ◽  
Active Site Residues ◽  
Content Type ◽  
And Function

ABSTRACTNew Delhi metallo-β-lactamase-1 (NDM-1) is expressed by various members ofEnterobacteriaceaeas a defense mechanism to hydrolyze β-lactam antibiotics. Despite various studies showing the significance of active-site residues in the catalytic mechanism, there is a paucity of reports addressing the role of non-active-site residues in the structure and function of NDM-1. In this study, we investigated the significance of non-active-site residue Trp-93 in the structure and function of NDM-1. We clonedblaNDM-1from anEnterobacter cloacaeclinical strain (EC-15) and introduced the mutation of Trp-93 to Ala (yielding the Trp93Ala mutant) by PCR-based site-directed mutagenesis. Proteins were expressed and purified to homogeneity by affinity chromatography. The MICs of the Trp93Ala mutant were reduced 4- to 8-fold for ampicillin, cefotaxime, ceftazidime, cefoxitin, imipenem, and meropenem. The poor hydrolytic activity of the Trp93Ala mutant was also reflected by its reduced catalytic efficiency. The overall catalytic efficiency of the Trp93Ala mutant was reduced by 40 to 55% (theKmwas reduced, while thekcatwas similar to that of wild-type NDM-1 [wtNDM-1]). Heat-induced denaturation showed that the ΔGDoandTmof Trp93Ala mutant were reduced by 1.8 kcal/mol and 4.8°C, respectively. Far-UV circular dichroism (CD) analysis showed that the α-helical content of the Trp93Ala mutant was reduced by 2.9%. The decrease in stability and catalytic efficiency of the Trp93Ala mutant was due to the loss of two hydrogen bonds with Ser-63 and Val-73 and hydrophobic interactions with Leu-65, Val-73, Gln-123, and Asp-124. The study provided insight into the role of non-active-site amino acid residues in the hydrolytic mechanism of NDM-1.

Role of the conserved active site residue tryptophan-24 of human dihydrofolate reductase as revealed by mutagenesis

Biochemistry ◽  
1991 ◽  
Vol 30 (5) ◽  
pp. 1432-1440 ◽  
Author(s):  
William A. Beard ◽  
James R. Appleman ◽  
Shaoming Huang ◽  
Tavner J. Delcamp ◽  
James H. Freisheim ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Active Site Residue ◽  
Human Dihydrofolate Reductase

Substitution of a non-active-site residue located on the T3 loop increased the catalytic efficiency of endo -polygalacturonases

2016 ◽  
Vol 51 (9) ◽  
pp. 1230-1238 ◽  
Author(s):  
Tao Tu ◽  
Xia Pan ◽  
Kun Meng ◽  
Huiying Luo ◽  
Rui Ma ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Active Site Residue

HPr: a model protein

1995 ◽  
Vol 73 (5-6) ◽  
pp. 219-222
Author(s):  
J. W. Anderson
Keyword(s):  
Structure Function ◽  
Active Center ◽  
Active Site ◽  
Central Component ◽  
Active Site Residue ◽  
Phosphotransferase System ◽  
Function Model ◽  
Model Protein

Histidine-containing protein (HPr) is a central component of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS). This brief review covers recent structure–function studies on the active center of this protein: the role of the active center residues in phosphotransfer; the residues contributing to the phosphohydrolysis properties of HPr; and the contribution residues in HPr make to the pKaof the transiently phosphorylated active-site residue, His 15. As well, the potential for HPr to be used as a model protein for studying problems not directly associated with its function in the PTS is discussed.Key words: phosphoenolpyruvate: sugar phosphotransferase system, histidine-containing protein, active center, structure–function, model protein.

Glutamic acid-65 is an essential residue for catalysis in Proteus mirabilis glutathione S-transferase B1-1

2002 ◽  
Vol 363 (1) ◽  
pp. 189-193 ◽  
Author(s):  
Nerino ALLOCATI ◽  
Michele MASULLI ◽  
Enrico CASALONE ◽  
Silvia SANTUCCI ◽  
Bartolo FAVALORO ◽  
...  
Keyword(s):  
Proteus Mirabilis ◽  
Active Site ◽  
Structural Integrity ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Cd Spectra ◽  
Glutathione S Transferase ◽  
Terminal Amino

The functional role of three conserved amino acid residues in Proteus mirabilis glutathione S-transferase B1-1 (PmGST B1-1) has been investigated by site-directed mutagenesis. Crystallographic analyses indicated that Glu65, Ser103 and Glu104 are in hydrogen-bonding distance of the N-terminal amino group of the γ-glutamyl moiety of the co-substrate, GSH. Glu65 was mutated to either aspartic acid or leucine, and Ser103 and Glu104 were both mutated to alanine. Glu65 mutants (Glu65→Asp and Glu65→Leu) lost all enzyme activity, and a drastic decrease in catalytic efficiency was observed for Ser103→Ala and Glu104→Ala mutants toward both 1-chloro-2,4-dinitrobenzene and GSH. On the other hand, all mutants displayed similar intrinsic fluorescence, CD spectra and thermal stability, indicating that the mutations did not affect the structural integrity of the enzyme. Taken together, these results indicate that Ser103 and Glu104 are significantly involved in the interaction with GSH at the active site of PmGST B1-1, whereas Glu65 is crucial for catalysis.

scholarly journals Structure-Based Mechanism for Early PLP-Mediated Steps of Rabbit Cytosolic Serine Hydroxymethyltransferase Reaction

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
Martino L. Di Salvo ◽  
J. Neel Scarsdale ◽  
Galina Kazanina ◽  
Roberto Contestabile ◽  
Verne Schirch ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Serine Hydroxymethyltransferase ◽  
Active Site Residue ◽  
Wild Type ◽  
Hydroxymethyl Group ◽  
New Energy ◽  
Mutant Forms

Serine hydroxymethyltransferase catalyzes the reversible interconversion of L-serine and glycine with transfer of one-carbon groups to and from tetrahydrofolate. Active site residue Thr254 is known to be involved in the transaldimination reaction, a crucial step in the catalytic mechanism of all pyridoxal 5′-phosphate- (PLP-) dependent enzymes, which determines binding of substrates and release of products. In order to better understand the role of Thr254, we have expressed, characterized, and determined the crystal structures of rabbit cytosolic serine hydroxymethyltransferase T254A and T254C mutant forms, in the absence and presence of substrates. These mutants accumulate a kinetically stablegem-diamine intermediate, and their crystal structures show differences in the active site with respect to wild type. The kinetic and crystallographic data acquired with mutant enzymes permit us to infer that conversion ofgem-diamine to external aldimine is significantly slowed because intermediates are trapped into an anomalous position by a misorientation of the PLP ring, and a new energy barrier hampers the transaldimination reaction. This barrier likely arises from the loss of the stabilizing hydrogen bond between the hydroxymethyl group of Thr254 and theε-amino group of active site Lys257, which stabilizes the external aldimine intermediate in wild type SHMTs.

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