Identification of active site residues by site-directed mutagenesis of delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B

Kim, Suhng Wook; Choi, Kwan Yong

doi:10.1128/jb.177.9.2602-2605.1995

Cited by 36 publications

(47 citation statements)

References 30 publications

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“…Protein Purification-Wild-type KSI from Pseudomonas putida isomerase (PI) and its mutant D38N-PI were overexpressed in Escherichia coli strain BL21 (DE3) and purified using a deoxycholate (DC) affinity column and a Superose 12 column (Amersham Pharmacia Biotech) as described (16). The D38N mutant PI gene was constructed by the site-directed mutagenesis method using a Muta-gene in vitro mutagenesis kit (Bio-Rad).…”

Section: Methodsmentioning

confidence: 99%

Detection of Large pK Perturbations of an Inhibitor and a Catalytic Group at an Enzyme Active Site, a Mechanistic Basis for Catalytic Power of Many Enzymes

Ha¹,

Kim²,

Lee³

et al. 2000

Journal of Biological Chemistry

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⌬ 5 -3-Ketosteroid isomerase catalyzes cleavage and formation of a C-H bond at a diffusion-controlled limit. By determining the crystal structures of the enzyme in complex with each of three different inhibitors and by nuclear magnetic resonance (NMR) spectroscopic investigation, we evidenced the ionization of a hydroxyl group (pK a ϳ16.5) of an inhibitor, which forms a low barrier hydrogen bond (LBHB) with a catalytic residue Tyr 14 (pK a ϳ11.5), and the protonation of the catalytic residue Asp 38 with pK a of ϳ4.5 at pH 6.7 in the interaction with a carboxylate group of an inhibitor. The perturbation of the pK a values in both cases arises from the formation of favorable interactions between inhibitors and catalytic residues. The results indicate that the pK a difference between catalytic residue and substrate can be significantly reduced in the active site environment as a result of the formation of energetically favorable interactions during the course of enzyme reactions. The reduction in the pK a difference should facilitate the abstraction of a proton and thereby eliminate a large fraction of activation energy in general acid/base enzyme reactions. The pK a perturbation provides a mechanistic ground for the fast reactivity of many enzymes and for the understanding of how some enzymes are able to extract a proton from a C-H group with a pK a value as high as ϳ30.Although the chemical nature of catalytic mechanisms is well understood for many enzymes, how enzymes are able to accelerate reaction rate by 10 8 -10 15 has remained an entirely unresolved issue. For example, the energetics of the seemingly simple heterolytic C-H bond cleavage is enigmatic, although it is a fundamental process found in a wide variety of biological reactions such as racemization, transamination, and isomerization (1, 2). In almost all cases, a proton is abstracted from a carbon adjacent to carbonyl, carboxylic acid, or carboxylate anion group by an active site residue because the ␣-protons are acidic by virtue of the resonance stabilization of the enolic intermediate. Despite the acidifying effect, the pK a values of the ␣-protons are typically much higher than the pK a value of a catalytic residue serving as a general base in the proton abstraction. The pK a values of the ␣-protons of most aldehydes, ketones, and thioesters lie between ϳ16 and 20, and pK a values of the ␣-protons of most carboxylate anions are Ն32, whereas those of active site bases are usually Ͻ7 (3). The unfavorable reverse proton transfer poses a large thermodynamic energy barrier the magnitude of which is a function of ⌬pK a , the difference in pK a between the proton donor and acceptor, and is given as 2.303RT⌬pK a (3), corresponding to 12-34 kcal/mol for the C-H bond cleavages. The required amount of energy has to be supplied by favorable interactions between catalytic residues and substrate in the course of catalysis, and/or the ⌬pK a must be significantly reduced in the active site milieu. The activation energy barrier may be entirely eliminated for reacti...

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Section: Methodsmentioning

confidence: 99%

Detection of Large pK Perturbations of an Inhibitor and a Catalytic Group at an Enzyme Active Site, a Mechanistic Basis for Catalytic Power of Many Enzymes

Ha¹,

Kim²,

Lee³

et al. 2000

Journal of Biological Chemistry

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“…The cultures were grown in 200 ml of Luria-Bertani medium containing 100 mg of ampicillin per liter for 16 to 20 h at 37°C, and the expression of mutant KSIs was induced by the addition of 0.75 mM isopropyl-␤-D-thiogalactopyranoside (IPTG) at the start of growth. The purification of the wild-type and mutant KSIs was carried out with affinity chromatography as the principal step according to procedures described previously (14). The KSI from C. testosteroni (5) was overexpressed and purified by a method similar to that described previously (12).…”

Section: Methodsmentioning

confidence: 99%

“…The KSI activity was determined spectrophotometrically at 248 nm in a system consisting of a 3-ml final solution containing 34 mM potassium phosphate (pH 7.0), 2.5 mM EDTA, 58.2 M 5-androstene-3,17-dione in methanol (1.7% final concentration) and enzyme as described previously (14). The reactions were initiated by the addition of enzyme, and all rates were corrected for the blank rates which were measured prior to the addition of enzyme.…”

Section: Methodsmentioning

confidence: 99%

“…This dimeric enzyme consists of two identical polypeptide chains of 131 amino acids each with a calculated molecular weight of 14,536. P. putida KSI is highly homologous to C. testosteroni KSI since the analysis of a primary sequence alignment of both proteins indicates that the critical active-site residues of C. testosteroni KSI, Asp-38 and Tyr-14, are conserved at Asp-40 and Tyr-16, respectively, in the P. putida KSI polypeptide (14,21). The catalytic mechanism of P. putida KSI was investigated to understand the roles that these conserved residues play by determining the activities of the mutant enzymes, and it has been proposed that Tyr-16 and Asp-40 are very similar to Tyr-14 and Asp-38 of C. testosteroni KSI in their roles as catalytic residues (14).…”

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confidence: 99%

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

et al. 1997

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In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by sitedirected mutagenesis. All cysteine mutations caused only a slight decrease in the k cat value, with no significant change of K m for the substrate. Even modification of the sulfhydryl group with 5,5-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an activesite-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by sitedirected mutagenesis. For D103A mutant KSI there was a significant decrease in the k cat value: the k cat of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k cat value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate.

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“…⌬ 5 -3-Ketosteroid isomerase (KSI, 1 EC 5.3.3.1) is one of the most proficient enzymes catalyzing an allylic isomerization reaction at a rate comparable to the diffusion-controlled limit by an intramolecular transfer of a proton (Scheme 1) (3-8). Although KSIs from two different bacterial sources, Comamonas testosteroni and Pseudomonas putida biotype B, share only 34% sequence identity, the catalytic residues Tyr-14, Asp-38, and Asp-99 (the residues are numbered according to C. testosteroni KSI) are well conserved in the two isomerases (9,10). Moreover, the overall three-dimensional structures are remarkably similar to each other, indicating that the two KSIs can share the same catalytic mechanism (11)(12)(13)(14).…”

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confidence: 99%

Origin of the Different pH Activity Profile in Two Homologous Ketosteroid Isomerases

Yun

Lee

Nam

et al. 2003

Journal of Biological Chemistry

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Two homologous ⌬5 -3-ketosteroid isomerases from Comamonas testosteroni (TI-WT) and Pseudomonas putida biotype B (PI-WT) exhibit different pH activity profiles. TI-WT loses activity below pH 5.0 due to the protonation of the conserved catalytic base, Asp-38, while PI-WT does not. Based on the structural analysis of PI-WT, the critical catalytic base, Asp-38, was found to form a hydrogen bond with the indole ring NH of Trp-116, which is homologously replaced with Phe-116 in TI-WT. To investigate the role of Trp-116, we prepared the F116W mutant of TI-WT (TI-F116W) and the W116F mutant of PI-WT (PI-W116F) and compared kinetic parameters of those mutants at different pH levels. PI-W116F exhibited significantly decreased catalytic activity at acidic pH like TI-WT, whereas TI-F116W maintained catalytic activity at acidic pH like PI-WT and increased the k cat /K m value by 2.5-to 4.7-fold compared with TI-WT at pH 3.8. The crystal structure of TI-F116W clearly showed that the indole ring NH of Trp-116 could form a hydrogen bond with the carboxyl oxygen of Asp-38 like that of PI-WT. The present results demonstrate that the activities of both PI-WT and TI-F116W at low pH were maintained by a tryptophan, which was able not only to lower the pK a value of the catalytic base but also to increase the substrate affinity. This is one example of the strategy nature can adopt to evolve the diversity of the catalytic function in the enzymes. Our results provide insight into deciphering the molecular evolution of the enzyme and creating novel enzymes by protein engineering.Isozymes carry out the same enzymatic reaction yet exhibit different properties such as substrate affinity, preference of different coenzyme, stability, optimal pH, etc. Even if the sequence differences of those isozymes are evident, the origin of different properties is mostly not clear at the molecular level. Identification of functional divergence on the mechanistic basis is important to understand how the enzyme as a molecular machine can be evolved in the biological system and to rationally modify the enzyme function with desired physical and catalytic properties (1). In the post-genomic era, protein sequences and high resolution protein structures are being accumulated rapidly, but the remarkable catalytic mechanism of enzymes as well as the catalytic strategies utilized by nature to evolve new catalytic functions remain to be understood at the molecular level (2). The understanding of the structural basis for different properties of isozymes and their catalytic diversity could provide the insights into not only discovering the properties of the enzymes from their protein sequences or structural data but also creating novel enzymes by protein engineering.⌬ 5 -3-Ketosteroid isomerase (KSI, 1 EC 5.3.3.1) is one of the most proficient enzymes catalyzing an allylic isomerization reaction at a rate comparable to the diffusion-controlled limit by an intramolecular transfer of a proton (Scheme 1) (3-8). Although KSIs from two different bacterial sources, Comamon...

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Identification of active site residues by site-directed mutagenesis of delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B

Abstract: In order to assess the roles of specific amino acid residues in the ⌬

Cited by 36 publications

References 30 publications

Detection of Large pK Perturbations of an Inhibitor and a Catalytic Group at an Enzyme Active Site, a Mechanistic Basis for Catalytic Power of Many Enzymes

Detection of Large pK Perturbations of an Inhibitor and a Catalytic Group at an Enzyme Active Site, a Mechanistic Basis for Catalytic Power of Many Enzymes

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

Origin of the Different pH Activity Profile in Two Homologous Ketosteroid Isomerases

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