scholarly journals Elimination of a reactive thiol group from the active site of chloramphenicol acetyltransferase

1990 ◽  
Vol 272 (2) ◽  
pp. 499-504 ◽  
Author(s):  
A Lewendon ◽  
W V Shaw
Keyword(s):  
Active Site ◽  
Thiol Group ◽  
Chloramphenicol Acetyltransferase ◽  
Side Chain ◽  
Kinetic Properties ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Hydrophobic Amino Acid ◽  
Nitrobenzoic Acid ◽  
Methionine Residue

1. The type III variant of chloramphenicol acetyltransferase (CATIII) is resistant to inactivation by ionizable modifying reagents such as 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and iodoacetate, whereas it is sensitive to inhibition by similar but uncharged reagents, including 4,4′-dithiodipyridine, methyl methanethiolsulphonate (MMTS) and iodoacetamide. The target for these thiol-modifying reagents has been postulated to be Cys-31. This residue is situated within a part of the chloramphenicol-binding site formed largely from the side chains of hydrophobic amino acid residues, which might be expected to discriminate against the access of ionized ligands to Cys-31. 2. The substitution of Cys-31 by alanine, serine, threonine or methionine yields an enzyme that is resistant to inactivation by thiol-specific reagents. Replacement of Cys-31 by alanine, serine or threonine results in increased Km values for chloramphenicol with only small changes in kcat.. In contrast, the Cys-31----Met substitution mainly affects kcat. values. Although the kcat. for chloramphenicol acetylation is decreased 13-fold compared with wild-type CAT, the kcat. for the acetyl-CoA hydrolysis reaction, which occurs in the absence of chloramphenicol, is increased 2.7-fold. 3. MMTS modification of cysteine residues results in an adduct (-CH2-S-S-CH3) that is structurally similar to the side chain of a methionine residue (-CH2-CH2-S-CH3). The kinetic properties of MMTS-modified CATIII closely resemble those of [Met31]CAT.

scholarly journals Site-directed mutagenesis of β-lactamase I: role of Glu-166

1994 ◽  
Vol 299 (3) ◽  
pp. 671-678 ◽  
Author(s):  
Y C Leung ◽  
C V Robinson ◽  
R T Aplin ◽  
S G Waley
Keyword(s):  
Active Site ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Kinetic Properties ◽  
Ionization Mass ◽  
Beta Lactamase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Important Residue

Two Glu-166 mutants of beta-lactamase I from Bacillus cereus 569/H were constructed: one with a lengthened side chain (E166Cmc, the S-carboxymethylcysteine mutant) and the other with the side chain shortened and made non-polar (E166A). Their kinetic properties were studied and compared with those of the wild-type and the E166D mutant (with a shortened side chain) previously made by Gibson, Christensen and Waley (1990) (Biochem. J. 272, 613-619). Surprisingly, with good penicillin substrates, Km, kcat. and kcat./Km of the two conservative mutants (E166Cmc and E166D) are similar to those of the non-conservative mutant E166A. Their kcat. values are 3000-fold lower than that of the wild-type enzyme, showing that Glu-166 is a very important residue. The acylenzyme intermediate of E166A and a good substrate, penicillin V, was trapped by acid-quench and observed by electrospray ionization mass spectrometry, suggesting that Glu-166 is more important in catalysing the deacylation step than the acylation step. The beta-lactamase I E166A mutant is about 200-fold more active than the Bacillus licheniformis E166A mutant with nitrocefin or 6 beta-furylacryloyl-amidopenicillanic acid as substrate. This suggested that other groups in the active site of the beta-lactamase I mutant may activate the catalytic water molecule for deacylation.

scholarly journals Gating Competence of Constitutively Open CLC-0 Mutants Revealed by the Interaction with a Small Organic Inhibitor

2003 ◽  
Vol 122 (3) ◽  
pp. 295-306 ◽  
Author(s):  
Sonia Traverso ◽  
Laura Elia ◽  
Michael Pusch
Keyword(s):  
Chloride Channels ◽  
Bound State ◽  
Side Chain ◽  
Pore Channel ◽  
Kinetic Properties ◽  
Wild Type ◽  
High Affinity ◽  
Critical Residue ◽  
Fast Gating

Opening of CLC chloride channels is coupled to the translocation of the permeant anion. From the recent structure determination of bacterial CLC proteins in the closed and open configuration, a glutamate residue was hypothesized to form part of the Cl−-sensitive gate. The negatively charged side-chain of the glutamate was suggested to occlude the permeation pathway in the closed state, while opening of a single protopore of the double-pore channel would reflect mainly a movement of this side-chain toward the extracellular pore vestibule, with little rearrangement of the rest of the channel. Here we show that mutating this critical residue (Glu166) in the prototype Torpedo CLC-0 to alanine, serine, or lysine leads to constitutively open channels, whereas a mutation to aspartate strongly slowed down opening. Furthermore, we investigated the interaction of the small organic channel blocker p-chlorophenoxy-acetic acid (CPA) with the mutants E166A and E166S. Both mutants were strongly inhibited by CPA at negative voltages with a >200-fold larger affinity than for wild-type CLC-0 (apparent KD at −140 mV ∼4 μM). A three-state linear model with an open state, a low-affinity and a high-affinity CPA-bound state can quantitatively describe steady-state and kinetic properties of the CPA block. The parameters of the model and additional mutagenesis suggest that the high-affinity CPA-bound state is similar to the closed configuration of the protopore gate of wild-type CLC-0. In the E166A mutant the glutamate side chain that occludes the permeation pathway is absent. Thus, if gating consists only in movement of this side-chain the mutant E166A should not be able to assume a closed conformation. It may thus be that fast gating in CLC-0 is more complex than anticipated from the bacterial structures.

scholarly journals Chemical modification of serine at the active site of penicillin acylase from Kluyvera citrophila

1991 ◽  
Vol 280 (3) ◽  
pp. 659-662 ◽  
Author(s):  
J Martín ◽  
A Slade ◽  
A Aitken ◽  
R Arche ◽  
R Virden
Keyword(s):  
Active Site ◽  
Tertiary Structure ◽  
Thiol Group ◽  
Iodoacetic Acid ◽  
Penicillin Acylase ◽  
Protein Product ◽  
Side Chain ◽  
Serine Residue ◽  
Conserved Sequence ◽  
Ester Substrate

The site of reaction of penicillin acylase from Kluyvera citrophila with the potent inhibitor phenylmethanesulphonyl fluoride was investigated by incubating the inactivated enzyme with thioacetic acid to convert the side chain of the putative active-site serine residue to that of cysteine. The protein product contained one thiol group, which was reactive towards 2,2′-dipyridyl disulphide and iodoacetic acid. Carboxymethylcysteine was identified as the N-terminal residue of the beta-subunit of the carboxy[3H]methylthiol-protein. No significant changes in tertiary structure were detected in the modified penicillin acylase using near-u.v. c.d. spectroscopy. However, the catalytic activity (kcat) with either an anilide or an ester substrate was decreased in the thiol-protein by a factor of more than 10(4). A comparison of sequences of apparently related acylases shows no other extensive regions of conserved sequence containing an invariant serine residue. The side chain of this residue is proposed as a candidate nucleophile in the formation of an acyl-enzyme during catalysis.

Structure of the Mutant E92K of [2Fe–2S] Ferredoxin I from Spinacia oleracea at 1.7 Å Resolution

1998 ◽  
Vol 54 (6) ◽  
pp. 1353-1358 ◽  
Author(s):  
Claudia Binda ◽  
Alessandro Coda ◽  
Alessandro Aliverti ◽  
Giuliana Zanetti ◽  
Andrea Mattevi
Keyword(s):  
Spinacia Oleracea ◽  
Crystal Packing ◽  
Side Chain ◽  
Molecular Replacement ◽  
Amino Acid Residues ◽  
Hydrogen Bond Network ◽  
Wild Type ◽  
Active Centre ◽  
Bond Network ◽  
Ferredoxin I

Ferredoxin I (Fd I) from Spinacia oleracea is composed of 97 amino-acid residues and a [2Fe–2S] cluster. The crystal structure of the E92K mutant of Fd I was solved by molecular replacement and refined to an R factor of 19.6% for 11755 reflections at 1.7 Å resolution. The overall structure and the active centre of spinach Fd is highly conserved with respect to ferredoxins of known structure. The E92K mutation appears to disturb a hydrogen-bond network which stabilizes the loop bearing the [2Fe–2S] cluster. This observation provides a rationale for the reduced electron-transfer efficiency displayed by the E92K mutant. Inspection of the crystal packing reveals that the side chain of Lys92 is engaged in an intermolecular interaction with Asp26 of a symmetry-related molecule. This feature may explain why only the mutant E92K and not wild-type Fd I could be successfully crystallized.

scholarly journals Roles of key active-site residues in flavocytochrome P450 BM3

1999 ◽  
Vol 339 (2) ◽  
pp. 371-379 ◽  
Author(s):  
Michael A. NOBLE ◽  
Caroline S. MILES ◽  
Stephen K. CHAPMAN ◽  
Dominikus A. LYSEK ◽  
Angela C. MACKAY ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Active Site ◽  
Side Chain ◽  
Acid Oxidation ◽  
Dodecanoic Acid ◽  
Wild Type ◽  
Active Site Residues ◽  
P450 Bm3

The effects of mutation of key active-site residues (Arg-47, Tyr-51, Phe-42 and Phe-87) in Bacillus megaterium flavocytochrome P450 BM3 were investigated. Kinetic studies on the oxidation of laurate and arachidonate showed that the side chain of Arg-47 contributes more significantly to stabilization of the fatty acid carboxylate than does that of Tyr-51 (kinetic parameters for oxidation of laurate: R47A mutant, Km 859 µM, kcat 3960 min-1; Y51F mutant, Km 432 µM, kcat 6140 min-1; wild-type, Km 288 µM, kcat 5140 min-1). A slightly increased kcat for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (ΔG‡) resulting from a smaller ΔG of substrate binding. The side chain of Phe-42 acts as a phenyl ‘cap ’ over the mouth of the substrate-binding channel. With mutant F42A, Km is massively increased and kcat is decreased for oxidation of both laurate (Km 2.08 mM, kcat 2450 min-1) and arachidonate (Km 34.9 µM, kcat 14620 min-1; compared with values of 4.7 µM and 17100 min-1 respectively for wild-type). Amino acid Phe-87 is critical for efficient catalysis. Mutants F87G and F87Y not only exhibit increased Km and decreased kcat values for fatty acid oxidation, but also undergo an irreversible conversion process from a ‘fast ’ to a ‘slow ’ rate of substrate turnover [for F87G (F87Y)-catalysed laurate oxidation: kcat ‘fast ’, 760 (1620) min-1; kcat ‘slow ’, 48.0 (44.6) min-1; kconv (rate of conversion from fast to slow form), 4.9 (23.8) min-1]. All mutants showed less than 10% uncoupling of NADPH oxidation from fatty acid oxidation. The rate of FMN-to-haem electron transfer was shown to become rate-limiting in all mutants analysed. For wild-type P450 BM3, the rate of FMN-to-haem electron transfer (8340 min-1) is twice the steady-state rate of oxidation (4100 min-1), indicating that other steps contribute to rate limitation. Active-site structures of the mutants were probed with the inhibitors 12-(imidazolyl)dodecanoic acid and 1-phenylimidazole. Mutant F87G binds 1-phenylimidazole > 10-fold more tightly than does the wild-type, whereas mutant Y51F binds the haem-co-ordinating fatty acid analogue 12-(imidazolyl)dodecanoic acid > 30-fold more tightly than wild-type.

scholarly journals His224 Alters the R2 Drug Binding Site and Phe218 Influences the Catalytic Efficiency of the Metallo-β-Lactamase VIM-7

2014 ◽  
Vol 58 (8) ◽  
pp. 4826-4836 ◽  
Author(s):  
Hanna-Kirsti S. Leiros ◽  
Susann Skagseth ◽  
Kine Susann Waade Edvardsen ◽  
Marit Sjo Lorentzen ◽  
Gro Elin Kjæreng Bjerga ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Active Site ◽  
Pathogenic Bacteria ◽  
Catalytic Efficiency ◽  
Drug Binding ◽  
Side Chain ◽  
Wild Type ◽  
Drug Binding Site ◽  
Chain Conformations ◽  
Positively Charged

ABSTRACTMetallo-β-lactamases (MBLs) are the causative mechanism for resistance to β-lactams, including carbapenems, in many Gram-negative pathogenic bacteria. One important family of MBLs is the Verona integron-encoded MBLs (VIM). In this study, the importance of residues Asp120, Phe218, and His224 in the most divergent VIM variant, VIM-7, was investigated to better understand the roles of these residues in VIM enzymes through mutations, enzyme kinetics, crystal structures, thermostability, and docking experiments. The tVIM-7-D120A mutant with a tobacco etch virus (TEV) cleavage site was enzymatically inactive, and its structure showed the presence of only the Zn1 ion. The mutant was less thermostable, with a melting temperature (Tm) of 48.5°C, compared to 55.3°C for the wild-type tVIM-7. In the F218Y mutant, a hydrogen bonding cluster was established involving residues Asn70, Asp84, and Arg121. The tVIM-7-F218Y mutant had enhanced activity compared to wild-type tVIM-7, and a slightly higherTm(57.1°C) was observed, most likely due to the hydrogen bonding cluster. Furthermore, the introduction of two additional hydrogen bonds adjacent to the active site in the tVIM-7-H224Y mutant gave a higher thermostability (Tm, 62.9°C) and increased enzymatic activity compared to those of the wild-type tVIM-7. Docking of ceftazidime in to the active site of tVIM-7, tVIM-7-H224Y, and VIM-7-F218Y revealed that the side-chain conformations of residue 224 and Arg228 in the L3 loop and Tyr67 in the L1 loop all influence possible substrate binding conformations. In conclusion, the residue composition of the L3 loop, as shown with the single H224Y mutation, is important for activity particularly toward the positively charged cephalosporins like cefepime and ceftazidime.

scholarly journals Role of methionine in the active site of α-galactosidase from Trichoderma reesei

1995 ◽  
Vol 308 (3) ◽  
pp. 955-964 ◽  
Author(s):  
A M Kachurin ◽  
A M Golubev ◽  
M M Geisow ◽  
O S Veselkina ◽  
L S Isaeva-Ivanova ◽  
...  
Keyword(s):  
Amino Acid ◽  
Trichoderma Reesei ◽  
Active Site ◽  
Fold Increase ◽  
Thiol Group ◽  
Competitive Inhibitor ◽  
Amino Acid Residues ◽  
Activity Curve ◽  
Oxidative Activation ◽  
Alpha Galactosidase

alpha-Galactosidase from Trichoderma reesei when treated with H2O2 shows a 12-fold increase in activity towards p-nitrophenyl alpha-D-galactopyranoside. A similar effect is produced by the treatment of alpha-galactosidase with other non-specific oxidants: NaIO4, KMnO4 and K4S4O8. In addition to the increase in activity, the Michaelis constant rises from 0.2 to 1.4 mM, the temperature coefficient decreases by a factor of 1.5 and the pH-activity curve falls off sharply with increasing pH. Galactose (a competitive inhibitor of alpha-galactosidase; Ki 0.09 mM for the native enzyme at pH 4.4) effectively inhibits oxidative activation of the enzyme, because the observed activity changes are related to oxidation of the catalytically important methionine in the active site. NMR measurements and amino acid analysis show that oxidation to methionine sulphoxide of one of five methionines is sufficient to activate alpha-galactosidase. Binding of galactose prevents this. Oxidative activation does not lead to conversion of other H2O2-sensitive amino acid residues, such as histidine, tyrosine, tryptophan and cysteine. The catalytically important cysteine thiol group is quantitatively titrated after protein oxidative activation. Further oxidation of methionines (up to four of five residues) can be achieved by increasing the oxidation time and/or by prior denaturation of the protein. Obviously, a methionine located in the active site of alpha-galactosidase is more accessible. The oxidative-activation phenomenon can be explained by a conformational change in the active site as a result of conversion of non-polar methionine into polar methionine sulphoxide.

scholarly journals Structural and Mutagenic Analysis of Metallo-β-Lactamase IMP-18

2016 ◽  
Vol 60 (9) ◽  
pp. 5521-5526 ◽  
Author(s):  
Takamitsu Furuyama ◽  
Haruka Nonomura ◽  
Yoshikazu Ishii ◽  
Nancy D. Hanson ◽  
Akiko Shimizu-Ibuka
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Kinetic Properties ◽  
Amino Acid Residues ◽  
Content Type ◽  
Zinc Metalloenzymes ◽  
Order Of Magnitude ◽  
Sandwich Configuration ◽  
The Relationship ◽  
Loop Conformation

ABSTRACTIMP-type metallo-β-lactamases (MBLs) are exogenous zinc metalloenzymes that hydrolyze a broad range of β-lactams, including carbapenems. Here we report the crystal structure of IMP-18, an MBL cloned fromPseudomonas aeruginosa, at 2.0-Å resolution. The overall structure of IMP-18 resembles that of IMP-1, with an αβ/βα “folded sandwich” configuration, but the loop that covers the active site has a distinct conformation. The relationship between IMP-18's loop conformation and its kinetic properties was investigated by replacing the amino acid residues that can affect the loop conformation (Lys44, Thr50, and Ile69) in IMP-18 with those occupying the corresponding positions in the well-described enzyme IMP-1. The replacement of Thr50 with Pro considerably modified IMP-18's kinetic properties, specifically those pertaining to meropenem, with thekcat/Kmvalue increased by an order of magnitude. The results indicate that this is a key residue that defines the kinetic properties of IMP-type β-lactamases.

Three factors that modulate the activity of class D β-lactamases and interfere with the post-translational carboxylation of Lys70

2010 ◽  
Vol 432 (3) ◽  
pp. 495-506 ◽  
Author(s):  
Lionel Vercheval ◽  
Cédric Bauvois ◽  
Alexandre di Paolo ◽  
Franck Borel ◽  
Jean-Luc Ferrer ◽  
...  
Keyword(s):  
Active Site ◽  
Chloride Ions ◽  
Side Chain ◽  
Wild Type ◽  
Post Translational Modification ◽  
Rate Limiting Step ◽  
Class D ◽  
The Individual ◽  
Rate Limiting

The activity of class D β-lactamases is dependent on Lys70 carboxylation in the active site. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D β-lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl–enzyme is the rate-limiting step for the wild-type OXA-10 β-lactamase.

Sign in / Sign up

Export Citation Format

Share Document