Structural insights into the effect of active-site mutation on the catalytic mechanism of carbonic anhydrase

Enzymes are catalysts of biological processes. Significant insight into their catalytic mechanisms has been obtained by relating site-directed mutagenesis studies to kinetic activity assays. However, revealing the detailed relationship between structural modifications and functional changes remains challenging owing to the lack of information on reaction intermediates and of a systematic way of connecting them to the measured kinetic parameters. Here, a systematic approach to investigate the effect of an active-site-residue mutation on a model enzyme, human carbonic anhydrase II (CA II), is described. Firstly, structural analysis is performed on the crystallographic intermediate states of native CA II and its V143I variant. The structural comparison shows that the binding affinities and configurations of the substrate (CO2) and product (HCO3 −) are altered in the V143I variant and the water network in the water-replenishment pathway is restructured, while the proton-transfer pathway remains mostly unaffected. This structural information is then used to estimate the modifications of the reaction rate constants and the corresponding free-energy profiles of CA II catalysis. Finally, the obtained results are used to reveal the effect of the V143I mutation on the measured kinetic parameters (k cat and k cat/K m) at the atomic level. It is believed that the systematic approach outlined in this study may be used as a template to unravel the structure–function relationships of many other biologically important enzymes.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj20020545 ◽

2002 ◽

Vol 367 (1) ◽

pp. 255-261 ◽

Cited By ~ 32

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.

Download Full-text

Active site of Zn2+-dependentsn-glycerol-1-phosphate dehydrogenase fromAeropyrum pernixK1

Archaea ◽

10.1155/2005/257264 ◽

2005 ◽

Vol 1 (5) ◽

pp. 311-317 ◽

Cited By ~ 8

Author(s):

Jin-Suk Han ◽

Kazuhiko Ishikawa

Keyword(s):

Active Site ◽

Catalytic Mechanism ◽

Structural Information ◽

Zinc Ion ◽

Site Directed Mutagenesis ◽

Glycerol Dehydrogenase ◽

Dihydroxyacetone Phosphate ◽

Wild Type ◽

Double Mutation ◽

Low Activity

The enzymesn-glycerol-1-phosphate dehydrogenase (Gro1PDH, EC 1.1.1.261) is key to the formation of the enantiomeric configuration of the glycerophosphate backbone (sn-glycerol-1-phosphate) of archaeal ether lipids. This enzyme catalyzes the reversible conversion between dihydroxyacetone phosphate and glycerol-1-phosphate. To date, no information about the active site and catalytic mechanism of this enzyme has been reported. Using the sequence and structural information for glycerol dehydrogenase, we constructed six mutants (D144N, D144A, D191N, H271A, H287A and D191N/H271A) of Gro1PDH fromAeropyrum pernixK1 and examined their characteristics to clarify the active site of this enzyme. The enzyme was found to be a zinc-dependent metalloenzyme, containing one zinc ion for every monomer protein that was essential for activity. Site-directed mutagenesis of D144 increased the activity of the enzyme. Mutants D144N and D144A exhibited low affinity for the substrates and higher activity than the wild type, but their affinity for the zinc ion was the same as that of the wild type. Mutants D191N, H271A and H287A had a low affinity for the zinc ion and a low activity compared with the wild type. The double mutation, D191N/ H271A, had no enzyme activity and bound no zinc. From these results, it was clarified that residues D191, H271 and H287 participate in the catalytic activity of the enzyme by binding the zinc ion, and that D144 has an effect on substrate binding. The structure of the active site of Gro1PDH fromA. pernixK1 seems to be similar to that of glycerol dehydrogenase, despite the differences in substrate specificity and biological role.

Download Full-text

Catalytic and structural contributions for glutathione-binding residues in a Delta class glutathione S-transferase

Biochemical Journal ◽

10.1042/bj20040697 ◽

2004 ◽

Vol 382 (2) ◽

pp. 751-757 ◽

Cited By ~ 23

Author(s):

Pakorn WINAYANUWATTIKUN ◽

Albert J. KETTERMAN

Keyword(s):

Binding Site ◽

Active Site ◽

Structural Integrity ◽

Catalytic Mechanism ◽

Tertiary Structure ◽

Site Directed Mutagenesis ◽

Active Site Residues ◽

Anopheles Dirus ◽

Steady State Kinetics ◽

Cellular Detoxification

Glutathione S-transferases (GSTs) are dimeric proteins that play a major role in cellular detoxification. The GSTs in mosquito Anopheles dirus species B, an important malaria vector in South East Asia, are of interest because they can play an important role in insecticide resistance. In the present study, we characterized the Anopheles dirus (Ad)GST D3-3 which is an alternatively spliced product of the adgst1AS1 gene. The data from the crystal structure of GST D3-3 shows that Ile-52, Glu-64, Ser-65, Arg-66 and Met-101 interact directly with glutathione. To study the active-site function of these residues, alanine substitution site-directed mutagenesis was performed resulting in five mutants: I52A (Ile-52→Ala), E64A, S65A, R66A and M101A. Interestingly, the E64A mutant was expressed in Escherichia coli in inclusion bodies, suggesting that this residue is involved with the tertiary structure or folding property of this enzyme. However, the I52A, S65A, R66A and M101A mutants were purified by glutathione affinity chromatography and the enzyme activity characterized. On the basis of steady-state kinetics, difference spectroscopy, unfolding and refolding studies, it was concluded that these residues: (1) contribute to the affinity of the GSH-binding site (‘G-site’) for GSH, (2) influence GSH thiol ionization, (3) participate in kcat regulation by affecting the rate-limiting step of the reaction, and in the case of Ile-52 and Arg-66, influenced structural integrity and/or folding of the enzyme. The structural perturbations from these mutants are probably transmitted to the hydrophobic-substrate-binding site (‘H-site’) through changes in active site topology or through effects on GSH orientation. Therefore these active site residues appear to contribute to various steps in the catalytic mechanism, as well as having an influence on the packing of the protein.

Download Full-text

Characterization of Self-Processing Activities and Substrate Specificities of Porcine Torovirus 3C-Like Protease

Journal of Virology ◽

10.1128/jvi.01282-20 ◽

2020 ◽

Vol 94 (20) ◽

Author(s):

Shangen Xu ◽

Junwei Zhou ◽

Yingjin Chen ◽

Xue Tong ◽

Zixin Wang ◽

...

Keyword(s):

Active Site ◽

Catalytic Mechanism ◽

Site Directed Mutagenesis ◽

Active Site Residues ◽

Hydrophobic Force ◽

Substrate Specificities ◽

Consensus Sequences ◽

Cleavage Activity ◽

Porcine Torovirus

ABSTRACT The 3C-like protease (3CLpro) of nidovirus plays an important role in viral replication and manipulation of host antiviral innate immunity, which makes it an ideal antiviral target. Here, we characterized that porcine torovirus (PToV; family Tobaniviridae, order Nidovirales) 3CLpro autocatalytically releases itself from the viral precursor protein by self-cleavage. Site-directed mutagenesis suggested that PToV 3CLpro, as a serine protease, employed His53 and Ser160 as the active-site residues. Interestingly, unlike most nidovirus 3CLpro, the P1 residue plays a less essential role in N-terminal self-cleavage of PToV 3CLpro. Substituting either P1 or P4 residue of substrate alone has little discernible effect on N-terminal cleavage. Notably, replacement of the two residues together completely blocks N-terminal cleavage, suggesting that N-terminal self-cleavage of PToV 3CLpro is synergistically affected by both P1 and P4 residues. Using a cyclized luciferase-based biosensor, we systematically scanned the polyproteins for cleavage sites and identified (FXXQ↓A/S) as the main consensus sequences. Subsequent homology modeling and biochemical experiments suggested that the protease formed putative pockets S1 and S4 between the substrate. Indeed, mutants of both predicted S1 (D159A, H174A) and S4 (P62G/L185G) pockets completely lost the ability of cleavage activity of PToV 3CLpro. In conclusion, the characterization of self-processing activities and substrate specificities of PToV 3CLpro will offer helpful information for the mechanism of nidovirus 3C-like proteinase’s substrate specificities and the rational development of the antinidovirus drugs. IMPORTANCE Currently, the active-site residues and substrate specificities of 3C-like protease (3CLpro) differ among nidoviruses, and the detailed catalytic mechanism remains largely unknown. Here, porcine torovirus (PToV) 3CLpro cleaves 12 sites in the polyproteins, including its N- and C-terminal self-processing sites. Unlike coronaviruses and arteriviruses, PToV 3CLpro employed His53 and Ser160 as the active-site residues that recognize a glutamine (Gln) at the P1 position. Surprisingly, mutations of P1-Gln impaired the C-terminal self-processing but did not affect N-terminal self-processing. The “noncanonical” substrate specificity for its N-terminal self-processing was attributed to the phenylalanine (Phe) residue at the P4 position in the N-terminal site. Furthermore, a double glycine (neutral) substitution at the putative P4-Phe-binding residues (P62G/L185G) abolished the cleavage activity of PToV 3CLpro suggested the potential hydrophobic force between the PToV 3CLpro and P4-Phe side chains.

Download Full-text

Site-Directed Mutagenesis of N5-Carboxyaminoimidazole Ribonucleotide Mutase (Class I PurE) in Bacillus anthracis

The Journal of Undergraduate Research at the University of Illinois at Chicago ◽

10.5210/jur.v5i1.7507 ◽

2011 ◽

Vol 5 (1) ◽

Author(s):

M. Silas ◽

N. Wolf ◽

L.W.M. Fung

Keyword(s):

Bacillus Anthracis ◽

Active Site ◽

De Novo ◽

Structural Information ◽

Site Directed Mutagenesis ◽

Class I ◽

Substrate Molecule ◽

Antibiotic Development ◽

Product Molecule ◽

Future Experimentation

Anthrax, the infection caused by the Gram-positive pathogen Bacillus anthracis (B. anthracis), is fatal if untreated, and some strains of B. anthracis have been found to be resistant to currently available antibiotics. The development of broad spectrum antibiotics is needed to treat the resistive strains. In antibiotic development, we have targeted B. anthracis Class I PurE enzyme (Ba PurE) as a unique and necessary enzyme in the de novo purine biosynthesis pathway, since the inactivation of this gene prevents B. anthraci growth in human serum, resulting in decreased bacterial proliferation. To identify inhibitors to $Ba$PurE, structural information on the substrate binding to its active site is needed. However, it is difficult to obtain crystals of Ba PurE with the substrate molecule in its binding site since upon binding to PurE, the substrate molecule is converted to the product molecule. An alternative approach is to create mutants of PurE that exhibit no enzymatic activity and do not convert the substrate to product, but still allow the substrate to bind to the active site. Then, the structure of mutant PurE with bound substrate can be obtained. We have identified a histidine residue at position 70 as the target of mutation to give an inactive enzyme. After successfully preparing the recombinant protein H70N, we have found that it exhibited no enzyme activity. This mutant will be useful in future experimentation to identify inhibitors of Ba PurE.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj3030357 ◽

1994 ◽

Vol 303 (2) ◽

pp. 357-362 ◽

Cited By ~ 29

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.

Download Full-text

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

Biochemical Journal ◽

10.1042/bj20011468 ◽

2002 ◽

Vol 365 (1) ◽

pp. 303-309 ◽

Cited By ~ 20

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.

Download Full-text

Streptomyces albus G serine β-lactamase. Probing of the catalytic mechanism via molecular modelling of mutant enzymes

Biochemical Journal ◽

10.1042/bj2820189 ◽

1992 ◽

Vol 282 (1) ◽

pp. 189-195 ◽

Cited By ~ 17

Author(s):

J Lamotte-Brasseur ◽

F Jacob-Dubuisson ◽

G Dive ◽

J M Frère ◽

J M Ghuysen

Keyword(s):

Hydrogen Bonding ◽

Active Site ◽

Catalytic Mechanism ◽

Site Directed Mutagenesis ◽

Network Configuration ◽

Beta Lactam ◽

Beta Lactamases ◽

Class A ◽

Streptomyces Albus ◽

Hydrogen Bonding Network

In previous studies, several amino acids of the active site of class A beta-lactamases have been modified by site-directed mutagenesis. On the basis of the catalytic mechanism proposed for the Streptomyces albus G beta-lactamase [Lamotte-Brasseur, Dive, Dideberg, Charlier, Frère & Ghuysen (1991) Biochem. J. 279, 213-221], the influence that these mutations exert on the hydrogen-bonding network of the active site has been analysed by molecular mechanics. The results satisfactorily explain the effects of the mutations on the kinetic parameters of the enzyme's activity towards a set of substrates. The present study also shows that, upon binding a properly structured beta-lactam compound, the impaired cavity of a mutant enzyme can readopt a functional hydrogen-bonding-network configuration.

Download Full-text