General Base Catalysis by the Phosphatidylcholine-Preferring Phospholipase C fromBacillus cereus:  The Role of Glu4 and Asp55†

Biochemistry ◽  
1998 ◽  
Vol 37 (16) ◽  
pp. 5755-5760 ◽  
Author(s):  
Stephen F. Martin ◽  
Paul J. Hergenrother
Keyword(s):  
Phospholipase C ◽  
Base Catalysis ◽  
General Base

Role of Electrophilic and General Base Catalysis in the Mechanism ofEscherichia coliUracil DNA Glycosylase†

Biochemistry ◽  
1999 ◽  
Vol 38 (37) ◽  
pp. 11866-11875 ◽  
Author(s):  
Alexander C. Drohat ◽  
Jayashree Jagadeesh ◽  
Eric Ferguson ◽  
James T. Stivers
Keyword(s):  
Base Catalysis ◽  
Dna Glycosylase ◽  
General Base

scholarly journals Structural and catalytic effects of an invariant purine substitution in the hammerhead ribozyme: implications for the mechanism of acid–base catalysis

2014 ◽  
Vol 70 (9) ◽  
pp. 2256-2263 ◽  
Author(s):  
Eric P. Schultz ◽  
Ernesto E. Vasquez ◽  
William G. Scott
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Hammerhead Ribozyme ◽  
Base Catalysis ◽  
Metal Ion Binding ◽  
Acid Base ◽  
Kinetic Measurements ◽  
Structural Role ◽  
General Base

The hammerhead ribozyme catalyzes RNA cleavageviaacid–base catalysis. Whether it does so by general acid–base catalysis, in which the RNA itself donates and abstracts protons in the transition state, as is typically assumed, or by specific acid–base catalysis, in which the RNA plays a structural role and proton transfer is mediated by active-site water molecules, is unknown. Previous biochemical and crystallographic experiments implicate an invariant purine in the active site, G12, as the general base. However, G12 may play a structural role consistent with specific base catalysis. To better understand the role of G12 in the mechanism of hammerhead catalysis, a 2.2 Å resolution crystal structure of a hammerhead ribozyme fromSchistosoma mansoniwith a purine substituted for G12 in the active site of the ribozyme was obtained. Comparison of this structure (PDB entry 3zd4), in which A12 is substituted for G, with three previously determined structures that now serve as important experimental controls, allows the identification of structural perturbations that are owing to the purine substitution itself. Kinetic measurements for G12 purine-substituted schistosomal hammerheads confirm a previously observed dependence of rate on the pKaof the substituted purine; in both cases inosine, which is similar to G in pKaand hydrogen-bonding properties, is unexpectedly inactive. Structural comparisons indicate that this may primarily be owing to the lack of the exocyclic 2-amino group in the G12A and G12I substitutions and its structural effect upon both the nucleotide base and phosphate of A9. The latter involves the perturbation of a previously identified and well characterized metal ion-binding site known to be catalytically important in both minimal and full-length hammerhead ribozyme sequences. The results permit it to be suggested that G12 plays an important role in stabilizing the active-site structure. This result, although not inconsistent with the potential role of G12 as a general base, indicates that an alternative hammerhead cleavage mechanism involving specific base catalysis may instead explain the observed rate dependence upon purine substitutions at G12. The crystallographic results, contrary to previous assumptions, therefore cannot be interpreted to favor the general base catalysis mecahnism over the specific base catalysis mechanism. Instead, both of these mutually exclusive mechanistic alternatives must be considered in light of the current structural and biochemical data.

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.

Stimulation of Ca(2+)-dependent exocytosis of the sperm acrosome by cAMP acting downstream of phospholipase A2

Reproduction ◽  
2000 ◽  
pp. 57-68 ◽  
Author(s):  
J Garde ◽  
ER Roldan
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Phosphodiesterase Inhibitors ◽  
Dibutyryl Camp ◽  
Camp Phosphodiesterase ◽  
Ram Sperm ◽  
Camp Formation ◽  
Stimulation Of

Spermatozoa undergo exocytosis in response to agonists that induce Ca2+ influx and, in turn, activation of phosphoinositidase C, phospholipase C, phospholipase A2, and cAMP formation. Since the role of cAMP downstream of Ca2+ influx is unknown, this study investigated whether cAMP modulates phospholipase C or phospholipase A2 using a ram sperm model stimulated with A23187 and Ca2+. Exposure to dibutyryl-cAMP, phosphodiesterase inhibitors or forskolin resulted in enhancement of exocytosis. However, the effect was not due to stimulation of phospholipase C or phospholipase A2: in spermatozoa prelabelled with [3H]palmitic acid or [14C]arachidonic acid, these reagents did not enhance [3H]diacylglycerol formation or [14C]arachidonic acid release. Spermatozoa were treated with the phospholipase A2 inhibitor aristolochic acid, and dibutyryl-cAMP to test whether cAMP acts downstream of phospholipase A2. Under these conditions, exocytosis did not occur in response to A23187 and Ca2+. However, inclusion of dibutyryl-cAMP and the phospholipase A2 metabolite lysophosphatidylcholine did result in exocytosis (at an extent similar to that seen when cells were treated with A23187/Ca2+ and without the inhibitor). Inclusion of lysophosphatidylcholine alone, without dibutyryl-cAMP, enhanced exocytosis to a lesser extent, demonstrating that cAMP requires a phospholipase A2 metabolite to stimulate the final stages of exocytosis. These results indicate that cAMP may act downstream of phospholipase A2, exerting a regulatory role in the exocytosis triggered by physiological agonists.

Kinetics of cyclization of S-ethoxycarbonylmethylisothiouronium chloride to 2-imino-4-thiazolidone

1979 ◽  
Vol 44 (3) ◽  
pp. 912-917 ◽  
Author(s):  
Vladimír Macháček ◽  
Said A. El-bahai ◽  
Vojeslav Štěrba
Keyword(s):  
Hydrochloric Acid ◽  
Dilute Hydrochloric Acid ◽  
Base Catalysis ◽  
General Base ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Kinetics Of Formation ◽  
Acid Catalyzed ◽  
Rate Limiting ◽  
Kinetics Of

Kinetics of formation of 2-imino-4-thiazolidone from S-ethoxycarbonylmethylisothiouronium chloride has been studied in aqueous buffers and dilute hydrochloric acid. The reaction is subject to general base catalysis, the β value being 0.65. Its rate limiting step consists in acid-catalyzed splitting off of ethoxide ion from dipolar tetrahedral intermediate. At pH < 2 formation of this intermediate becomes rate-limiting; rate constant of its formation is 2 . 104 s-1.

Evidence for general base catalysis by protic solvents in a kinetic study of alcoholyses and hydrolyses of 1-(phenoxycarbonyl)pyridinium ions under both solvolytic and non-solvolytic conditions

2009 ◽  
Vol 74 (1) ◽  
pp. 43-55 ◽  
Author(s):  
Dennis N. Kevill ◽  
Byoung-Chun Park ◽  
Jin Burm Kyong
Keyword(s):  
Second Order ◽  
Base Catalysis ◽  
Substitution Reactions ◽  
Third Order ◽  
Methoxy Derivative ◽  
First Order ◽  
General Base ◽  
Protic Solvents ◽  
Kinetics Of ◽  
Pyridinium Ions

The kinetics of nucleophilic substitution reactions of 1-(phenoxycarbonyl)pyridinium ions, prepared with the essentially non-nucleophilic/non-basic fluoroborate as the counterion, have been studied using up to 1.60 M methanol in acetonitrile as solvent and under solvolytic conditions in 2,2,2-trifluoroethan-1-ol (TFE) and its mixtures with water. Under the non- solvolytic conditions, the parent and three pyridine-ring-substituted derivatives were studied. Both second-order (first-order in methanol) and third-order (second-order in methanol) kinetic contributions were observed. In the solvolysis studies, since solvent ionizing power values were almost constant over the range of aqueous TFE studied, a Grunwald–Winstein equation treatment of the specific rates of solvolysis for the parent and the 4-methoxy derivative could be carried out in terms of variations in solvent nucleophilicity, and an appreciable sensitivity to changes in solvent nucleophilicity was found.

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