Acid−Base Catalysis in the Extradiol Catechol Dioxygenase Reaction Mechanism:  Site-Directed Mutagenesis of His-115 and His-179 inEscherichia coli2,3-Dihydroxyphenylpropionate 1,2-Dioxygenase (MhpB)†,‡

Biochemistry ◽  
2004 ◽  
Vol 43 (42) ◽  
pp. 13390-13396 ◽  
Author(s):  
Sharon Mendel ◽  
Andrew Arndt ◽  
Timothy D. H. Bugg
Keyword(s):  
Reaction Mechanism ◽  
Base Catalysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Acid Base ◽  
Catechol Dioxygenase

scholarly journals Site-directed mutagenesis at aspartate and glutamate residues of xylanase from Bacillus pumilus

1992 ◽  
Vol 288 (1) ◽  
pp. 117-121 ◽  
Author(s):  
E P Ko ◽  
H Akatsuka ◽  
H Moriyama ◽  
A Shinmyo ◽  
Y Hata ◽  
...  
Keyword(s):  
Amino Acids ◽  
Reaction Mechanism ◽  
Amino Acid ◽  
Bacillus Pumilus ◽  
Specific Activity ◽  
Sequence Similarity ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Sequence Similarity ◽  
Directed Mutagenesis

To elucidate the reaction mechanism of xylanase, the identification of amino acids essential for its catalysis is of importance. Studies have indicated the possibility that the reaction mechanism of xylanase is similar to that of hen's egg lysozyme, which involves acidic amino acid residues. On the basis of this assumption, together with the three-dimensional structure of Bacillus pumilus xylanase and its amino acid sequence similarity to other xylanases of different origins, three acidic amino acids, namely Asp-21, Glu-93 and Glu-182, were selected for site-directed mutagenesis. The Asp residue was altered to either Ser or Glu, and the Glu residues to Ser or Asp. The purified mutant xylanases D21E, D21S, E93D, E93S, E182D and E182S showed single protein bands of about 26 kDa on SDS/PAGE. C.d. spectra of these mutant enzymes show no effect on the secondary structure of xylanase, except that of D21E, which shows a little variation. Furthermore, mutations of Glu-93 and Glu-182 resulted in a drastic decrease in the specific activity of xylanase as compared with mutation of Asp-21. On the basis of these results we propose that Glu-93 and Glu-182 are the best candidates for the essential catalytic residues of xylanase.

scholarly journals Insights into the reaction mechanism of Escherichia coli agmatinase by site-directed mutagenesis and molecular modelling

2002 ◽  
Vol 269 (22) ◽  
pp. 5522-5526 ◽  
Author(s):  
Mónica Salas ◽  
Rolando Rodríguez ◽  
Nelia López ◽  
Elena Uribe ◽  
Vasthi López ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Reaction Mechanism ◽  
Molecular Modelling ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Acid–base catalysis in Leuconostoc mesenteroides sucrose phosphorylase probed by site-directed mutagenesis and detailed kinetic comparison of wild-type and Glu237→Gln mutant enzymes

2007 ◽  
Vol 403 (3) ◽  
pp. 441-449 ◽  
Author(s):  
Alexandra Schwarz ◽  
Lothar Brecker ◽  
Bernd Nidetzky
Keyword(s):  
Leuconostoc Mesenteroides ◽  
Ph Dependence ◽  
Base Catalysis ◽  
Glycoside Hydrolases ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Acid Base ◽  
Sucrose Phosphorylase ◽  
Nmr Studies ◽  
Enzymatic Function

The role of acid–base catalysis in the two-step enzymatic mechanism of α-retaining glucosyl transfer by Leuconostoc mesenteroides sucrose phosphorylase has been examined through site-directed replacement of the putative catalytic Glu237 and detailed comparison of purified wild-type and Glu237→Gln mutant enzymes using steady-state kinetics. Reactions with substrates requiring Brønsted catalytic assistance for glucosylation or deglucosylation were selectively slowed at the respective step, about 105-fold, in E237Q. Azide, acetate and formate but not halides restored catalytic activity up to 300-fold in E237Q under conditions in which the deglucosylation step was rate-determining, and promoted production of the corresponding α-glucosides. In situ proton NMR studies of the chemical rescue of E237Q by acetate and formate revealed that enzymatically formed α-glucose 1-esters decomposed spontaneously via acyl group migration and hydrolysis. Using pH profiles of kcat/Km, the pH dependences of kinetically isolated glucosylation and deglucosylation steps were analysed for wild-type and E237Q. Glucosylation of the wild-type proceeded optimally above and below apparent pKa values of about 5.6 and 7.2 respectively whereas deglucosylation was dependent on the apparent single ionization of a group of pKa≈5.8 that must be deprotonated for reaction. Glucosylation of E237Q was slowed below apparent pKa≈6.0 but had lost the high pH dependence of the wild-type. Deglucosylation of E237Q was pH-independent. The results allow unequivocal assignment of Glu237 as the catalytic acid–base of sucrose phosphorylase. They support a mechanism in which the pKa of Glu237 cycles between ≈7.2 in free enzyme and ≈5.8 in glucosyl enzyme intermediate, ensuring optimal participation of the glutamate residue side chain at each step in catalysis. Enzyme deglucosylation to an anionic nucleophile took place with Glu237 protonated or unprotonated. The results delineate how conserved active-site groups of retaining glycoside hydrolases can accommodate enzymatic function of a phosphorylase.

scholarly journals Site-directed mutagenesis reveals a novel catalytic mechanism of Mycobacterium tuberculosis alkylhydroperoxidase C

2002 ◽  
Vol 367 (1) ◽  
pp. 255-261 ◽  
Author(s):  
Radha CHAUHAN ◽  
Shekhar C. MANDE
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Reaction Mechanism ◽  
Kinetic Parameters ◽  
Active Site ◽  
Double Mutant ◽  
Catalytic Mechanism ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Resistant Strains

Mycobacterium tuberculosis alkylhydroperoxidase C (AhpC) belongs to the peroxiredoxin family, but unusually contains three cysteine residues in its active site. It is overexpressed in isoniazid-resistant strains of M. tuberculosis. We demonstrate that AhpC is capable of acting as a general antioxidant by protecting a range of substrates including supercoiled DNA. Active-site Cys to Ala mutants show that all three cysteine residues are important for activity. Cys-61 plays a central role in activity and Cys-174 also appears to be crucial. Interestingly, the C174A mutant is inactive, but double mutant C174/176A shows significant revertant activity. Kinetic parameters indicate that the C176A mutant is active, although much less efficient. We suggest that M. tuberculosis AhpC therefore belongs to a novel peroxiredoxin family and might follow a unique disulphide-relay reaction mechanism.

scholarly journals Study of the role of the highly conserved residues Arg9 and Arg64 in the catalytic function of human N-acetyltransferases NAT1 and NAT2 by site-directed mutagenesis

1997 ◽  
Vol 323 (1) ◽  
pp. 207-215 ◽  
Author(s):  
Claudine DELOMÉNIE ◽  
Geoffrey H. GOODFELLOW ◽  
Rajagopal KRISHNAMOORTHY ◽  
Denis M. GRANT ◽  
Jean-Marie DUPRET
Keyword(s):  
Ph Dependence ◽  
Conformational Stability ◽  
Base Catalysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Base Catalysts ◽  
General Base ◽  
Catalytic Function ◽  
Transition State Stabilization

The arylamine N-acetyltransferases (NATs) NAT1 and NAT2 are responsible for the biotransformation of many arylamine and hydroxylamine xenobiotics. It has been proposed that NATs may act through a cysteine-linked acetyl-enzyme intermediate in a general base catalysis involving a highly conserved arginine residue such as Arg64. To investigate this possibility, we used site-directed mutagenesis and expression of recombinant human NAT1 and NAT2 in Escherichia coli. Sequence comparison with NATs from other species indicated that Arg9 and Arg64 are the only invariant basic residues. Either mutation of the presumed catalytic Cys68 residue or the simultaneous mutation of Arg9 and Arg64 to Ala produced proteins with undetectable enzyme activity. NAT1 or NAT2 singly substituted at Arg9 or Arg64 with Ala, Met, Gln or Lys exhibited unaltered Km values for arylamine acceptor substrates, but a marked loss of activity and stability. Finally, double replacement of Arg9/Arg64 with lysine in NAT1 altered the Km for arylamine substrates (decreased by 8-14-fold) and for acetyl-CoA (elevated 5-fold), and modified the pH-dependence of activity. Thus, through their positively charged side chains, Arg9 and Arg64 seem to contribute to the conformational stability of NAT1 and NAT2 rather than acting as general base catalysts. Our results also support a mechanism in which Arg9 and Arg64 are involved in substrate binding and transition-state stabilization of NAT1.

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