scholarly journals Contribution of a buried aspartate residue towards the catalytic efficiency and structural stability of Bacillus stearothermophilus lactate dehydrogenase

1994 ◽  
Vol 300 (2) ◽  
pp. 491-499 ◽  
Author(s):  
T J Nobbs ◽  
A Cortés ◽  
J L Gelpi ◽  
J J Holbrook ◽  
T Atkinson ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Lactate Dehydrogenase ◽  
Active Site ◽  
Bacillus Stearothermophilus ◽  
Mutant Enzyme ◽  
Hydroxyl Group ◽  
Side Chain ◽  
General Effect ◽  
Carboxylate Group ◽  
Wild Type

The X-ray structure of lactate dehydrogenase (LDH) shows the side-chain carboxylate group of Asp-143 to be buried in the hydrophobic interior of the enzyme, where it makes hydrogen-bonding interactions with both the side-chain hydroxyl group of Ser-273 and the main-chain amide group of His-195. This is an unusual environment for a carboxylate side-chain as hydrogen bonding normally occurs with water molecules at the surface of the protein. A charged hydrogen-bonding interaction in the interior of a protein would be expected to be much stronger than a similar interaction on the solvent-exposed exterior. In this respect the side-chain carboxylate group of Asp-143 appears to be important for maintaining tertiary structure by providing a common linkage point between three discontinuous elements of the secondary structure, alpha 1F, beta K and the beta-turn joining beta G and beta H. The contribution of the Asp-143 side-chain to the structure and function of Bacillus stearothermophilus LDH was assessed by creating a mutant enzyme containing Asn-143. The decreased thermal stability of both unactivated and fructose-1,6-diphosphate (Fru-1,6-P2)-activated forms of the mutant enzyme support a structural role for Asp-143. Furthermore, the difference in stability of the wild-type and mutant enzymes in guanidinium chloride suggested that the carboxylate group of Asp-143 contributes at least 22 kJ/mol to the conformational stability of the wild-type enzyme. However, there was no alteration in the amount of accessible tryptophan fluorescence in the mutant enzyme, indicating that the mutation caused a structural weakness rather than a gross conformational change. Comparison of the wild-type and mutant enzyme steady-state parameters for various 2-keto acid substrates showed the mutation to have a general effect on catalysis, with an average difference in binding energy of 11 kJ/mol for the transition-state complexes. The different effects of pH and Fru-1,6-P2 on the wild-type and mutant enzymes also confirmed a perturbation of the catalytic centre in the mutant enzyme. As the side-chain of Asp-143 is not sufficiently close to the active site to be directly involved in catalysis or substrate binding it is proposed that the effects on catalysis shown by the mutant enzyme are induced either by a structural change or by charge imbalance at the active site.(ABSTRACT TRUNCATED AT 400 WORDS)

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 Re-design of Saccharomyces cerevisiae flavocytochrome b2: introduction of l-mandelate dehydrogenase activity

1998 ◽  
Vol 333 (1) ◽  
pp. 117-120 ◽  
Author(s):  
Rhona SINCLAIR ◽  
Graeme A. REID ◽  
Stephen K. CHAPMAN
Keyword(s):  
Lactate Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Active Site ◽  
Mutant Enzyme ◽  
Activity Levels ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Activity Increase ◽  
Double Mutation ◽  
Flavocytochrome B2

Flavocytochrome b2 from Saccharomyces cerevisiaeis an l-lactate dehydrogenase which exhibits only barely detectable activity levels towards another 2-hydroxyacid, l-mandelate. Using protein engineering methods we have altered the active site of flavocytochrome b2 and successfully introduced substantial mandelate dehydrogenase activity into the enzyme. Changes to Ala-198 and Leu-230 have significant effects on the ability of the enzyme to utilize l-mandelate as a substrate. The double mutation of Ala-198 → Gly and Leu-230 → Ala results in an enzyme with a kcat value (25 °C) with l-mandelate of 8.5 s-1, which represents an increase of greater than 400-fold over the wild-type enzyme. Perhaps more significantly, the mutant enzyme has a catalytic efficiency (as judged by kcat/Km values) that is 6-fold higher with l-mandelate than it is with l-lactate. Closer examination of the X-ray structure of S. cerevisiae flavocytochrome b2 led us to conclude that one of the haem propionate groups might interfere with the binding of l-mandelate at the active site of the enzyme. To test this idea, the activity with l-mandelate of the independently expressed flavodehydrogenase domain (FDH), was examined and found to be higher than that seen with the wild-type enzyme. In addition, the double mutation of Ala-198 → Gly and Leu-230 → Ala introduced into FDH produced the greatest mandelate dehydrogenase activity increase, with a kcat value more than 700-fold greater than that seen with the wild-type holoenzyme. In addition, the enzyme efficiency (kcat/Km) of this mutant enzyme was more than 20-fold greater with l-mandelate than with l-lactate. We have therefore succeeded in constructing an enzyme which is now a better mandelate dehydrogenase than a lactate dehydrogenase.

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.

An investigation of the contribution made by the carboxylate group of an active site histidine-aspartate couple to binding and catalysis in lactate dehydrogenase

Biochemistry ◽  
1988 ◽  
Vol 27 (5) ◽  
pp. 1617-1622 ◽  
Author(s):  
Anthony R. Clarke ◽  
Helen M. Wilks ◽  
David A. Barstow ◽  
Tony Atkinson ◽  
William N. Chia ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Active Site ◽  
Carboxylate Group ◽  
Active Site Histidine

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.

scholarly journals Kinetic and stereochemical comparison of wild-type and active-site K145Q mutant enzyme of bacterial D-amino acid transaminase.

1993 ◽  
Vol 268 (10) ◽  
pp. 6932-6938
Author(s):  
M.B. Bhatia ◽  
S. Futaki ◽  
H. Ueno ◽  
J.M. Manning ◽  
D. Ringe ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Mutant Enzyme ◽  
Wild Type

scholarly journals Identification of a Long-range Protein Network That Modulates Active Site Dynamics in Extremophilic Alcohol Dehydrogenases

2013 ◽  
Vol 288 (20) ◽  
pp. 14087-14097 ◽  
Author(s):  
Zachary D. Nagel ◽  
Shujian Cun ◽  
Judith P. Klinman
Keyword(s):  
Long Range ◽  
Active Site ◽  
Bacillus Stearothermophilus ◽  
Isotope Effects ◽  
Thermostable Enzyme ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Aromatic Side Chain ◽  
Alcohol Dehydrogenases ◽  
Rate Determining Step

A tetrameric thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (ht-ADH) has been mutated at an aromatic side chain in the active site (Trp-87). The ht-W87A mutation results in a loss of the Arrhenius break seen at 30 °C for the wild-type enzyme and an increase in cold lability that is attributed to destabilization of the active tetrameric form. Kinetic isotope effects (KIEs) are nearly temperature-independent over the experimental temperature range, and similar in magnitude to those measured above 30 °C for the wild-type enzyme. This suggests that the rigidification in the wild-type enzyme below 30 °C does not occur for ht-W87A. A mutation at the dimer-dimer interface in a thermolabile psychrophilic homologue of ht-ADH, ps-A25Y, leads to a more thermostable enzyme and a change in the rate-determining step at low temperature. The reciprocal mutation in ht-ADH, ht-Y25A, results in kinetic behavior similar to that of W87A. Collectively, the results indicate that flexibility at the active site is intimately connected to a subunit interaction 20 Å away. The convex Arrhenius curves previously reported for ht-ADH (Kohen, A., Cannio, R., Bartolucci, S., and Klinman, J. P. (1999) Nature 399, 496–499) are proposed to arise, at least in part, from a change in subunit interactions that rigidifies the substrate-binding domain below 30 °C, and impedes the ability of the enzyme to sample the catalytically relevant conformational landscape. These results implicate an evolutionarily conserved, long-range network of dynamical communication that controls C-H activation in the prokaryotic alcohol dehydrogenases.

scholarly journals The mutation Lys234His yields a class A β-lactamase with a novel pH-dependence

1991 ◽  
Vol 278 (3) ◽  
pp. 673-678 ◽  
Author(s):  
J Brannigan ◽  
A Matagne ◽  
F Jacob ◽  
C Damblon ◽  
B Joris ◽  
...  
Keyword(s):  
Active Site ◽  
Positive Charge ◽  
Ph Dependence ◽  
Side Chain ◽  
Beta Lactamase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Beta Lactamases ◽  
Class A ◽  
Transition State Stabilization

The lysine-234 residue is highly conserved in beta-lactamases and in nearly all active-site-serine penicillin-recognizing enzymes. Its replacement by a histidine residue in the Streptomyces albus G class A beta-lactamase yielded an enzyme the pH-dependence of which was characterized by the appearance of a novel pK, which could be attributed to the newly introduced residue. At low pH, the kcat, value for benzylpenicillin was as high as 50% of that of the wild-type enzyme, demonstrating that an efficient active site was maintained. Both kcat. and kcat/Km dramatically decreased above pH 6 but the decrease in kcat./Km could not be attributed to larger Km values. Thus a positive charge on the side chain of residue 234 appears to be more essential for transition-state stabilization than for initial recognition of the substrate ground state.

scholarly journals The stereospecificity of α-chymotrypsin

1968 ◽  
Vol 108 (4) ◽  
pp. 561-569 ◽  
Author(s):  
D. W. Ingles ◽  
J. R. Knowles
Keyword(s):  
Hydrogen Bonding ◽  
Active Site ◽  
High Energy ◽  
Side Chain ◽  
Acceptor Site ◽  
Amino Acid Side Chain ◽  
Hydrogen Bond Acceptor ◽  
Group 3 ◽  
Free Energy Of Binding ◽  
Hydrogen Bonding Site

1. The rates of deacylation of acyl-α-chymotrypsins in which the hydrogen-bonding capacity of the acylamino group of the substrate has been systematically removed were measured. 2. The ratio of deacylation rates of l- and d-acyl-enzymes is found to depend largely on the existence in the substrate of an amido –NH– group. 3. The data presented agree with the postulate that the stereospecificity of α-chymotrypsin is exercised in catalytic rather than binding steps, and that the active site of the enzyme presents three loci to the substrate: the site containing the catalytic functionalities (including serine-195), the hydrophobic area for amino acid side-chain binding, and a hydrogen-bond acceptor site for acylamino group binding. 4. It is noted that, though the hydrogen-bonding site is crucial for the stereospecificity, the free energy of binding of substrates and inhibitors is dominated by the hydrophobic interaction. 5. It is tentatively proposed that α-chymotrypsin selects a high-energy conformation of the substrate when the latter binds at the enzyme's active site.

Threonine 246 at the active site of the L-lactate dehydrogenase of Bacillus stearothermophilus is important for catalysis but not for substrate binding

Biochemistry ◽  
1993 ◽  
Vol 32 (47) ◽  
pp. 12730-12735 ◽  
Author(s):  
Roman Sakowicz ◽  
Helmut K. W. Kallwass ◽  
Wendy Parris ◽  
Cyril M. Kay ◽  
J. Bryan Jones ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Active Site ◽  
Bacillus Stearothermophilus ◽  
Substrate Binding
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