Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B

Kim, Suhng Wook; Joo, Sung Hyun; Choi, Gildon; Cho, Hyun Soo; Oh, Byung Ha; Choi, Kwan Yong

doi:10.1128/jb.179.24.7742-7747.1997

Cited by 23 publications

(28 citation statements)

References 37 publications

(35 reference statements)

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“…The predicted values of log(k cat ͞k cat(WT) ) are in good agreement with the values measured for PI (32)(33)(34)(35) and TI (25-28, 31) ( Table 1). Given that the highly homologous three-dimensional (tertiary and quaternary) structure and the active-site environ- Enzymatic reaction scheme of KSI.…”

Section: Resultssupporting

confidence: 82%

“…2C), the barrier for proton transfer is found to be very small in our calculation, and so the proton transfers in the double-welltype potential alone cannot explain the drastic lowering of the activation barrier. Indeed, case g, wherein the double-well-like characteristics of the potential almost disappear with little Table 1 seven model systems (a-g) Calculated relative reactivities were compared with the PI and TI experimental values (26,(31)(32)(33)(34)(35). The experimental activation barrier at TS1 of WT is estimated to be 10Ϸ11 kcal͞mol by Pollack and coworkers (46).…”

Section: Fig 2 Calculated Energy Profiles (⌬E) (A); Interoxygen Dismentioning

confidence: 99%

“…1), with diffusion-controlled reactivity, and serves as a paradigm for fast enzymatic enolization involving carbanions (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). Enzyme reactions associated with carbanion intermediates responsible for isomerization reaction and carbon-carbon bond formation͞cleavage are vital to metabolism of living organisms.…”

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confidence: 99%

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Catalytic role of enzymes: Short strong H-bond-induced partial proton shuttles and charge redistributions

Kim

Oh²,

Lee³

2000

Proc. Natl. Acad. Sci. U.S.A.

104

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A two-step reaction mechanism (catalyzed alternatively by acid and base) with partial proton shuttles and charge redistributions promoted by short strong H bonds (SSHBs) (playing a dual role as an amphi-acid͞base catalyst) is proposed to explain the enormous rate enhancement observed in enzymatic reactions involving carbanion intermediates. The SSHBs in the two-step reactions are found to be responsible for enhancing enzymesubstrate interactions in favor of the transition state structure over that of reactant. The detailed quantum theoretical studies of ketosteroid isomerase provide evidence of assisting roles of SSHB in enzymatic activity. The understanding of the two-step reaction mechanism would be a useful aid in designing novel functional enzymes and abzymes.A n understanding of how enzymes enhance the rate of reactions is essential for investigating the biological role of enzymes and designing new enzymes (1-3). In this context, enzyme-substrate interactions and enzyme preorganization have been invoked to explain the rate enhancement in favor of substrate transition state (TS) structures (4-14). In enzymesubstrate interactions, low barrier H bonds characterized by short and strong H bonds (SSHB) have been considered responsible for drastic enhancement in H-bond energies (4)(5)(6)(7)(8)(9)(10)(11)15). This concept has been introduced to explain the fast reactivities observed in various enzymes (4). Alternative explanations like preorganization in favor of the TS structures (mainly by electrostatic interactions) have also been proposed (12, 13), and the issue has been highly debated (9-24). In the present study, we attempt to resolve this issue by elucidating the origin of the catalytic role in ketosteroid isomerase (KSI), which is representative of this class of enzymes. We have carried out quantum theoretical calculations on the active sites of KSI to compare the experimental x-ray structures, NMR chemical shifts, and kinetic reaction rates for various mutants. We find evidence that the SSHB [driven by preorganized reaction environment in favor of the TS structure over the reactant structure, or enzymesubstrate complex (ES)] promotes both partial proton shuttles and (electronic) charge redistributions in a two-step mechanism as the role of an amphi-acid͞base catalyst, and hence eventually leads to a drastic lowering of the activation barrier in the catalytic reaction.KSI is one of the most proficient enzymes, catalyzing the isomerization of a variety of ⌬ 5 -3-ketosteroids to ⌬ 4 -3-ketosteroids (i.e., promoting an allylic rearrangement involving intramolecular proton transfer via a dienolic intermediate) (Fig. 1), with diffusion-controlled reactivity, and serves as a paradigm for fast enzymatic enolization involving carbanions (25-35). Enzyme reactions associated with carbanion intermediates responsible for isomerization reaction and carbon-carbon bond formation͞cleavage are vital to metabolism of living organisms.The important catalytic residues in Pseudomonas testosteroni KSI (TI) and Pseudomonas putid...

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Section: Resultssupporting

confidence: 82%

Section: Fig 2 Calculated Energy Profiles (⌬E) (A); Interoxygen Dismentioning

confidence: 99%

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Catalytic role of enzymes: Short strong H-bond-induced partial proton shuttles and charge redistributions

Kim

Oh²,

Lee³

2000

Proc. Natl. Acad. Sci. U.S.A.

104

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“…Thiocyanate labeling of KSI was accomplished by engineering single cysteine mutations at each of the above positions in the cysteine-free variant C69S/C81S/ C97S to ensure unique labeling. The Cys residues at positions 69, 81, and 97 are located on the enzyme's surface, and their removal has a negligible effect on catalysis (38).…”

Section: Resultsmentioning

confidence: 99%

Quantitative, directional measurement of electric field heterogeneity in the active site of ketosteroid isomerase

Fafarman¹,

Sigala²,

Schwans³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

216

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Understanding the electrostatic forces and features within highly heterogeneous, anisotropic, and chemically complex enzyme active sites and their connection to biological catalysis remains a longstanding challenge, in part due to the paucity of incisive experimental probes of electrostatic properties within proteins. To quantitatively assess the landscape of electrostatic fields at discrete locations and orientations within an enzyme active site, we have incorporated site-specific thiocyanate vibrational probes into multiple positions within bacterial ketosteroid isomerase. A battery of X-ray crystallographic, vibrational Stark spectroscopy, and NMR studies revealed electrostatic field heterogeneity of 8 MV∕cm between active site probe locations and widely differing sensitivities of discrete probes to common electrostatic perturbations from mutation, ligand binding, and pH changes. Electrostatic calculations based on active site ionization states assigned by literature precedent and computational pK a prediction were unable to quantitatively account for the observed vibrational band shifts. However, electrostatic models of the D40N mutant gave qualitative agreement with the observed vibrational effects when an unusual ionization of an active site tyrosine with a pK a near 7 was included. UV-absorbance and 13 C NMR experiments confirmed the presence of a tyrosinate in the active site, in agreement with electrostatic models. This work provides the most direct measure of the heterogeneous and anisotropic nature of the electrostatic environment within an enzyme active site, and these measurements provide incisive benchmarks for further developing accurate computational models and a foundation for future tests of electrostatics in enzymatic catalysis. E xtensive structural studies of enzymes have revealed that biological catalysis occurs within sequestered active site crevices that solvate reacting substrates with an anisotropic and chemically complex constellation of charged, polar, and hydrophobic groups. But beyond visualization of the chemical composition and architecture of active sites permitted by the rich library of available protein structures, our understanding of the electrostatic nature and properties of this highly heterogeneous environment and its role in molecular recognition and catalysis is largely based on simulations. Computations using three-dimensional structures routinely provide electrostatic potentials, as in Fig. 1A. These potentials have been used to identify and characterize protein-protein and protein-ligand binding sites, and interaction energies derived from these calculations have suggested key contributions from the electrostatic environment to enzymatic rate enhancement and specificity (1-5). However, even the most sophisticated computational methods have multiple approximations and limitations (6-8) and, most importantly, have not been rigorously evaluated through cycles of nontrivial computational predictions and experimental tests. Incisive and quantitative experimental measures o...

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“…The membrane was blocked in a solution containing phosphate-buffered saline (PBS) (4 mM KH 2 PO 4 , 16 mM Na 2 HPO 4 , 115 mM NaCl), 5 Covalent modification with DTNB or DEPC. A 0.75-ml portion of CzcD (170 mg ⅐ liter Ϫ1 ) was modified with 0.5 mM dithionitrobenzoic acid (DTNB) at 23°C for 40 min as described previously (11). Formation of thionitrobenzoic acid was monitored over time at 412 nm by using an extinction coefficient of 14,000 cm Ϫ1 ⅐ M Ϫ1 .…”

Section: Methodsmentioning

confidence: 99%

Characteristics of Zinc Transport by Two Bacterial Cation Diffusion Facilitators from Ralstonia metallidurans CH34 and Escherichia coli

et al. 2004

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CzcD from Ralstonia metallidurans and ZitB from Escherichia coli are prototypes of bacterial members of the cation diffusion facilitator (CDF) protein family. Expression of the czcD gene in an E. coli mutant strain devoid of zitB and the gene for the zinc-transporting P-type ATPase zntA rendered this strain more zinc resistant and caused decreased accumulation of zinc. CzcD, purified as an amino-terminal streptavidin-tagged protein, . Conserved amino acyl residues that might be involved in binding and transport of zinc were mutated in CzcD and/or ZitB, and the influence on Zn 2؉ resistance was studied. Charged or polar amino acyl residues that were located within or adjacent to membrane-spanning regions of the proteins were essential for the full function of the proteins. Probably, these amino acyl residues constituted a pathway required for export of the heavy metal cations or for import of counter-flowing protons.The cation diffusion facilitators (CDF) (T.C.2.A.4.1.1) (26) are a family of metal transport proteins found in a variety of organisms (16,17,19,23). In contrast to other protein families, such as P-type ATPases or ABC transporters (26), all CDF proteins characterized to date transport only metals, and the majority are involved in Zn 2ϩ transport (3,16,23). One of the first two CDF identified was the CzcD protein (15, 18) from the gram-negative bacterium Ralstonia metallidurans strain CH34 (previously Alcaligenes eutrophus [5]). This transporter is part of a cobalt-zinc-cadmium resistance system (Czc) and decreases the intracellular zinc concentration (1). The CzcD protein is composed of a hydrophobic, membrane-bound domain consisting of about 200 amino acid residues with probably six transmembrane ␣-helices and a 115-amino-acid hydrophilic domain located in the cytoplasm (1).Escherichia coli detoxifies excess Zn 2ϩ by using ZntA, a P-type ATPase, and the CDF protein ZitB (6), which is closely related to CzcD. Previously, site-directed mutagenesis was used to identify amino acyl residues H53, H159, D163, and D186 of ZitB as essential residues (12). These residues were located within predicted transmembrane domains (TMs) of this protein. In this study we compared a variety of additional mutations in ZitB and CzcD to better understand functional aspects of zinc binding and efflux. ZitB-dependent 65 Zn 2ϩ transport into everted membrane vesicles was driven by the proton motive force (PMF). These kinetics suggest that this protein is mainly responsible for zinc homeostasis under most physiological conditions. MATERIALS AND METHODSBacterial strains, growth conditions, and plasmid construction. The media used to cultivate E. coli strains W3110 (wild type) (7), GG48 (⌬zitB::Cm zntA::Km) (6), and GR362 (⌬zntA::Km ⌬zitB ⌬zupT ⌬znuABC ⌬zntB::Cm) (8) were Tris-buffered mineral salts medium (13) containing 2 g of glucose liter Ϫ1 plus 1 g of yeast extract liter Ϫ1 (TGY), the same medium with 2 g of glycerol liter Ϫ1 plus 3 g of Casamino Acids liter Ϫ1 (TGC), and Luria-Bertani broth (LB) (27). Solid Tris-buffered m...

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Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B

Cited by 23 publications

References 37 publications

Catalytic role of enzymes: Short strong H-bond-induced partial proton shuttles and charge redistributions

Catalytic role of enzymes: Short strong H-bond-induced partial proton shuttles and charge redistributions

Quantitative, directional measurement of electric field heterogeneity in the active site of ketosteroid isomerase

Characteristics of Zinc Transport by Two Bacterial Cation Diffusion Facilitators from Ralstonia metallidurans CH34 and Escherichia coli

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