Specific activation of a tyrosine → glycine mutant of Δ5-3-ketosteroid isomerase by phenols

Brooks, Bob; Benisek, William F.

doi:10.1016/s0006-291x(05)80036-1

Cited by 17 publications

(18 citation statements)

References 23 publications

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“…Since their report, this "chemical rescue" approach has been successfully applied to a number of enzymes to identify catalytic residues and to establish structure-activity relationships. The rescuer molecules used are amines for lysine-mutated enzymes (14 -16), guanidines for arginine-mutated enzymes (17,18), phenol for a tyrosine-mutated enzyme (19), azide or formate for a glutamate-mutated enzyme (20), and imidazole for histidinemutated enzymes (21,22). In this study, we have demonstrated by combination of chemical rescue studies and isotope effect analysis of the amine-assisted reactions catalyzed by K39A BSAR that Lys 39 of the wild-type BSAR probably mediates not only transaldimination but also ␣-hydrogen abstraction from the substrate.…”

Section: Resultsmentioning

confidence: 99%

Role of Lysine 39 of Alanine Racemase from Bacillus stearothermophilus That Binds Pyridoxal 5′-Phosphate

Watababe

Kurokawa

Yoshimura

et al. 1999

Journal of Biological Chemistry

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The lysine residue binding with the cofactor pyridoxal 5-phosphate (PLP) plays an important role in catalysis, such as in the transaldimination and abstraction of ␣-hydrogen from a substrate amino acid in PLP-dependent enzymes. We studied the role of Lys 39 of alanine racemase (EC 5.1.1.1) from Bacillus stearothermophilus, the PLP-binding residue of the enzyme, by replacing it site-specifically with alanine and characterizing the resultant K39A mutant enzyme. The mutant enzyme turned out to be inherently inactive, but gained an activity as high as about 0.1% of that of the wild-type enzyme upon addition of 0.2 M methylamine. The amineassisted activity of the mutant enzyme depended on the pK a values and molecular volumes of the alkylamines used. A strong kinetic isotope effect was observed when ␣-deuterated D-alanine was used as a substrate in the methylamine-assisted reaction, but little effect was observed using its antipode. In marked contrast, only Lenantiomer of alanine showed a solvent isotope effect in deuterium oxide in the methylamine-assisted reaction. These results suggest that methylamine serves as a base not only to abstract the ␣-hydrogen from D-alanine but also to transfer a proton from water to the ␣-position of the deprotonated (achiral) intermediate to form D-alanine. Therefore, the exogenous amine can be regarded as a functional group fully representing Lys 39 of the wild-type enzyme. Lys 39 of the wild-type enzyme probably acts as the base catalyst specific to the D-enantiomer of alanine. Another residue specific to the L-enantiomer in the wild-type enzyme is kept intact in the K39A mutant.

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Section: Resultsmentioning

confidence: 99%

Role of Lysine 39 of Alanine Racemase from Bacillus stearothermophilus That Binds Pyridoxal 5′-Phosphate

Watababe

Kurokawa

Yoshimura

et al. 1999

Journal of Biological Chemistry

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“…Plasmids encoding pKSI [ 129] and tKSI [ 130], which contain KSI inserted between the EcoRI and HinDIII sites of the pKK223–3 plasmid for the enzyme from P. putida and C. testosteroni, respectively, were gifts from Kwan Yong Choi and Ralph Pollack.…”

Section: Methodsmentioning

confidence: 99%

Testing Electrostatic Complementarity in Enzyme Catalysis: Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole

et al. 2006

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A longstanding proposal in enzymology is that enzymes are electrostatically and geometrically complementary to the transition states of the reactions they catalyze and that this complementarity contributes to catalysis. Experimental evaluation of this contribution, however, has been difficult. We have systematically dissected the potential contribution to catalysis from electrostatic complementarity in ketosteroid isomerase. Phenolates, analogs of the transition state and reaction intermediate, bind and accept two hydrogen bonds in an active site oxyanion hole. The binding of substituted phenolates of constant molecular shape but increasing p K a models the charge accumulation in the oxyanion hole during the enzymatic reaction. As charge localization increases, the NMR chemical shifts of protons involved in oxyanion hole hydrogen bonds increase by 0.50–0.76 ppm/p K a unit, suggesting a bond shortening of ˜0.02 Å/p K a unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates (ΔΔG = −0.2 kcal/mol/p K a unit). The small effect of increased charge localization on affinity occurs despite the shortening of the hydrogen bonds and a large favorable change in binding enthalpy (ΔΔH = −2.0 kcal/mol/p K a unit). This shallow dependence of binding affinity suggests that electrostatic complementarity in the oxyanion hole makes at most a modest contribution to catalysis of ˜300-fold. We propose that geometrical complementarity between the oxyanion hole hydrogen-bond donors and the transition state oxyanion provides a significant catalytic contribution, and suggest that KSI, like other enzymes, achieves its catalytic prowess through a combination of modest contributions from several mechanisms rather than from a single dominant contribution.

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“…Phillips et al (1992) showed that guanidine, methylguanidine, or ethylguanidine restored activity to an argininesubstituted carboxypeptidase mutant. Phenols or acetate have been employed to rescue mutant enzymes with a substituted tyrosine (Brooks & Benisek, 1992) or aspartic acid (Perona et al, 1994) residue. In each of these cases, the chemical rescue agent contains the same functional group as that of the substituted amino acid residue.…”

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confidence: 99%

Chemical Rescue by Guanidine Derivatives of an Arginine-Substituted Site-Directed Mutant of Escherichia coli Ornithine Transcarbamylase

Rynkiewicz

Seaton

1996

Biochemistry

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Escherichia coli ornithine transcarbamylase (OTCase) catalyzes the production of L-citrulline and phosphate from carbamyl phosphate and L-ornithine in L-arginine biosynthesis. We show that exogenous guanidines can restore activity to (chemically rescue) a catalytically-impaired site-directed mutant OTCase, R57G, in which glycine replaces an an active site arginine. The best rescue agent is guanidine hydrochloride, which enhances the rate of the mutant 2000-fold. The turnover number for the guanidine-rescued R57G mutant is 10% that of wild-type. The addition of guanidine to the R57G mutant has little effect on KMCP values, and the rescue effect is therefore attributed principally to an increase in kcat. Other compounds were screened as potential rescue agents, but rate enhancement is highly selective for guanidines. Not all guanidines show large increases in kcat. For a comparative series that includes guanidine and alkylguanidines, substituent size is inversely related to kcat. Brønsted analysis of guanidines with varying pKa values indicates that a partial positive charge is implicated in rescue, consistent with the proposed role of arginine 57 in catalysis. In UV difference and 31P-NMR spectra, carbamyl phosphate-induced effects associated with wild-type OTCase are observed in the R57G mutant only in the presence of guanidine. The kinetic mechanism of the mutant is random in the presence or absence of guanidine, in contrast to the sequential ordered mechanism of the wild-type enzyme. Thus, chemical rescue of R57G by guanidine hydrochloride restores many but not all wild-type properties to the mutant enzyme.

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Specific activation of a tyrosine → glycine mutant of Δ5-3-ketosteroid isomerase by phenols

Cited by 17 publications

References 23 publications

Role of Lysine 39 of Alanine Racemase from Bacillus stearothermophilus That Binds Pyridoxal 5′-Phosphate

Role of Lysine 39 of Alanine Racemase from Bacillus stearothermophilus That Binds Pyridoxal 5′-Phosphate

Testing Electrostatic Complementarity in Enzyme Catalysis: Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole

Chemical Rescue by Guanidine Derivatives of an Arginine-Substituted Site-Directed Mutant of Escherichia coli Ornithine Transcarbamylase

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