Insights into the Catalytic Mechanism and Active-Site Environment of Comamonas Testosteroni .DELTA.5-3-Ketosteroid Isomerase as Revealed by Site-Directed Mutagenesis of the Catalytic Base Aspartate-38

Biochemistry ◽  
1995 ◽  
Vol 34 (43) ◽  
pp. 14245-14253 ◽  
Author(s):  
Christopher M. Holman ◽  
William F. Benisek
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Comamonas Testosteroni ◽  
Ketosteroid Isomerase

scholarly journals Site-directed mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia coli

1994 ◽  
Vol 303 (2) ◽  
pp. 357-362 ◽  
Author(s):  
M P G van der Linden ◽  
L de Haan ◽  
O Dideberg ◽  
W Keck
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Binding Capacity ◽  
Binding Protein ◽  
Complete Loss ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Active Site Residues

Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser44), Lys47, Ser110-Gly-Asn, Asp175 and Lys213-Thr-Gly were identified as the residues making up the conserved boxes of this protein family. To determine the role of these residues, they were replaced using site-directed mutagenesis. The mutant proteins were assayed for their penicillin-binding capacity and DD-carboxypeptidase activity. The Ser44Cys and the Ser44Gly mutants showed a complete loss of both penicillin-binding capacity and DD-carboxypeptidase activity. The Lys47Arg mutant also lost its DD-carboxypeptidase activity but was able to bind and hydrolyse penicillin, albeit at a considerably reduced rate. Mutants in the Ser110-Gly-Asn fingerprint were affected in both acylation and deacylation upon reaction with penicillin and lost their DD-carboxypeptidase activity with the exception of Asn112Ser and Asn112Thr. The Asp175Asn mutant showed wild-type penicillin-binding but a complete loss of DD-carboxypeptidase activity. Mutants of Lys213 lost both penicillin-binding and DD-carboxypeptidase activity except for Lys213His, which still bound penicillin with a k+2/K' of 0.2% of the wild-type value. Mutation of His216 and Thr217 also had a strong effect on DD-carboxypeptidase activity. Thr217Ser and Thr217Ala showed augmented hydrolysis rates for the penicillin acyl-enzyme. This study reveals the residues in the conserved fingerprints to be very important for both DD-carboxypeptidase activity and penicillin-binding, and confirms them to play crucial roles in catalysis.

scholarly journals Role of αArg145 and βArg263 in the active site of penicillin acylase of Escherichia coli

2002 ◽  
Vol 365 (1) ◽  
pp. 303-309 ◽  
Author(s):  
Wynand B.L. ALKEMA ◽  
Antoon K. PRINS ◽  
Erik de VRIES ◽  
Dick B. JANSSEN
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Penicillin Acylase ◽  
Precursor Protein ◽  
Site Directed Mutagenesis ◽  
Penicillin G ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Arginine Residues

The active site of penicillin acylase of Escherichia coli contains two conserved arginine residues. The function of these arginines, αArg145 and βArg263, was studied by site-directed mutagenesis and kinetic analysis of the mutant enzymes. The mutants αArg145→Leu (αArg145Leu), αArg145Cys and αArg145Lys were normally processed and exported to the periplasm, whereas expression of the mutants βArg263Leu, βArg263Asn and βArg263Lys yielded large amounts of precursor protein in the periplasm, indicating that βArg263 is crucial for efficient processing of the enzyme. Either modification of both arginine residues by 2,3-butanedione or replacement by site-directed mutagenesis yielded enzymes with a decreased specificity (kcat/Km) for 2-nitro-5-[(phenylacetyl)amino]benzoic acid, indicating that both residues are important in catalysis. Compared with the wild type, the αArg145 mutants exhibited a 3–6-fold-increased preference for 6-aminopenicillanic acid as the deacylating nucleophile compared with water. Analysis of the steady-state parameters of these mutants for the hydrolysis of penicillin G and phenylacetamide indicated that destabilization of the Michaelis—Menten complex accounts for the improved activity with β-lactam substrates. Analysis of pH—activity profiles of wild-type enzyme and the βArg263Lys mutant showed that βArg263 has to be positively charged for catalysis, but is not involved in substrate binding. The results provide an insight into the catalytic mechanism of penicillin acylase, in which αArg145 is involved in binding of β-lactam substrates and βArg263 is important both for stabilizing the transition state in the reaction and for correct processing of the precursor protein.

Probing the catalytic mechanism of bovine CD38/NAD+glycohydrolase by site directed mutagenesis of key active site residues

2014 ◽  
Vol 1844 (7) ◽  
pp. 1317-1331 ◽  
Author(s):  
Isabelle Kuhn ◽  
Esther Kellenberger ◽  
Céline Cakir-Kiefer ◽  
Hélène Muller-Steffner ◽  
Francis Schuber
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Active Site Residues ◽  
Nad Glycohydrolase

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 The active site and substrate-binding mode of 1-aminocyclopropane-1-carboxylate oxidase determined by site-directed mutagenesis and comparative modelling studies

2004 ◽  
Vol 380 (2) ◽  
pp. 339-346 ◽  
Author(s):  
Young Sam SEO ◽  
Ahrim YOO ◽  
Jinwon JUNG ◽  
Soon-Kee SUNG ◽  
Dae Ryook YANG ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Binding Mode ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Directed Mutagenesis ◽  
Comparative Modelling

The active site and substrate-binding mode of MD-ACO1 (Malus domestica Borkh. 1-aminocyclopropane-1-carboxylate oxidase) have been determined using site-directed mutagenesis and comparative modelling methods. The MD-ACO1 protein folds into a compact jelly-roll motif comprised of eight α-helices, 12 β-strands and several long loops. The active site is well defined as a wide cleft near the C-terminus. The co-substrate ascorbate is located in cofactor Fe2+-binding pocket, the so-called ‘2-His-1-carboxylate facial triad’. In addition, our results reveal that Arg244 and Ser246 are involved in generating the reaction product during enzyme catalysis. The structure agrees well with the biochemical and site-directed mutagenesis results. The three-dimensional structure together with the steady-state kinetics of both the wild-type and mutant MD-ACO1 proteins reveal how the substrate specificity of MD-ACO1 is involved in the catalytic mechanism, providing insights into understanding the fruit ripening process at atomic resolution.

β-Amyrin biosynthesis: catalytic mechanism and substrate recognition

2017 ◽  
Vol 15 (14) ◽  
pp. 2869-2891 ◽  
Author(s):  
Tsutomu Hoshino
Keyword(s):  
Catalytic Mechanism ◽  
Substrate Recognition ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
The Past ◽  
Substrate Analog ◽  
Analog Experiments

In the past five years, there have been remarkable advances in the study of β-amyrin synthase. This review outlines the catalytic mechanism and substrate recognition in β-amyrin biosynthesis, which have been attained by the site-directed mutagenesis and substrate analog experiments.

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