Enzymic and Nonenzymic Polarizations of .alpha.,.beta.-Unsaturated Ketosteroids and Phenolic Steroids. Implications for the Roles of Hydrogen Bonding in the Catalytic Mechanism of .DELTA.5-3-Ketosteroid Isomerase

Zhao, Qinjian; Mildvan, Albert S.; Talalay, Paul

doi:10.1021/bi00002a006

Cited by 27 publications

(37 citation statements)

References 27 publications

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“…This latter mechanism may be similar to that observed for the acid-catalyzed hydrolysis of glucose 1-phosphate (40). Precedence for Tyr hydroxyls participating as general acids in reaction mechanisms has been obtained for the ketosteroid isomerase of Pseudomonas testosteroni (41) and the tyrosine phenol-lyase of Citrobacter freundii (42). The role of a general acid in PRT catalysis has also been proposed for the highly conserved Lys 103 of OPRTs, since mutation of this residue in the S. typhimurium enzyme caused a 1000-fold decrease in the k cat value of the enzyme without affecting the K m values for either the forward or reverse reaction (39).…”

Section: -Fold (28)supporting

confidence: 66%

The Conserved Serine-Tyrosine Dipeptide in Leishmania donovani Hypoxanthine-guanine Phosphoribosyltransferase Is Essential for Catalytic Activity

Jardim

Ullman

1997

Journal of Biological Chemistry

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Protozoan parasites constitute a devastating public health and socioeconomic burden in the tropical and subtropical regions of the world. In the absence of effective vaccines, empirically derived drugs are the only means available to treat parasitic infections. However, the current arsenal of antiparasitic drugs is far from ideal, as these agents are moderately to highly toxic, possibly mutagenic and/or carcinogenic, and require protracted treatment regimens. The control of parasitic diseases has also been complicated by the emergence of drugresistant strains (1), further underscoring the need for novel antiparasitic agents. Rational development of new drugs that selectively target protozoan pathogens requires the exploitation of biochemical or metabolic differences between parasite and host. As protozoan parasites, unlike mammalian cells, are auxotrophic for purines and consequently rely on purine appropriation from the host for survival and proliferation (2), the purine salvage pathway has stimulated considerable interest as a paradigm for antiparasitic drug development. One enzyme that is central to purine acquisition in many parasites (2) is hypoxanthine-guanine phosphoribosyltransferase (HGPRT) 1 which catalyzes the phosphoribosylpyrophosphate (PRPP)-dependent phosphoribosylation of hypoxanthine and guanine to the nucleotide level. In some parasites, HGPRT also recognizes xanthine as a substrate (3) and is, therefore, termed a hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGX-PRT). Genes and cDNAs encoding HG(X)PRT (HG(X)PRT) from a number of protozoan parasites have now been cloned, sequenced, and overexpressed in Escherichia coli, and the recombinant HG(X)PRT enzymes have been purified and characterized kinetically (3). Multisequence alignments demonstrate several short regions of amino acid homology among HG(X)-PRT family members that are flanked by much longer regions without significant sequence similarity. The crystal structures for the product-bound form of the human HGPRT (4) and the Tritrichomonas foetus HGXPRT (5) have revealed, however, a common core framework within the tertiary structure that is conserved among other members of the phosphoribosyltransferase (PRT) family (6 -8). These structures have established functional roles for all of the conserved regions in HG(X)PRT proteins, except one motif that is located on a flexible loop distal to the active site. This flexible loop encompasses a SerTyr dipeptide that is found in all HG(X)PRTs for which sequence is available. The recently determined crystal structures of the HGXPRT from Toxoplasma gondii in the absence of ligand and in the presence of product have demonstrated that this flexible loop in the apoenzyme is shifted toward the active

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Section: -Fold (28)supporting

confidence: 66%

The Conserved Serine-Tyrosine Dipeptide in Leishmania donovani Hypoxanthine-guanine Phosphoribosyltransferase Is Essential for Catalytic Activity

Jardim

Ullman

1997

Journal of Biological Chemistry

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“…Titrations were carried out by sequential additions of 3 l of the inhibitor solutions at several different concentrations with a total volume between 30 and 40 l. Before and after the addition of each inhibitor, the emission spectrum was scanned from 300 to 400 nm with an excitation wavelength at 290 nm. After correction of spectral changes caused by the dilution, the fluorescence of the enzyme at 334 nm for each inhibitor concentration was used to calculate the K d values as reported previously (21). The nonlinear least squares fitting in the calculation was performed with the program Kaleidagraph Version 2.6 (Abelbeck Software).…”

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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“…Although the existence of an LBHB between Tyr-14 and the dienolate intermediate on the isomerase has been suspected (6)(7)(8)(9)(10)(11), direct observation of this LBHB has been elusive. The participation of an LBHB in the mechanism of isomerase would resolve a perplexing question concerning the large and unfavorable difference in pKa values between the 3-carbonyl group of the substrate (pKa = -7) and that of the general acid, Tyr-14 (pKa = 11.6) (7).…”

mentioning

confidence: 99%

“…However, the exchange mechanism differs for the model system since specific-base catalysis is detected by the pH-dependence of the exchange rate over the limited pH range (10.0 to 12.2) that could be investigated, and by a significant entropic contribution to the kinetic barrier (-TASt = 4 kcal/mol), which is consistent with a second order process. Thus, an LBHB can exist even with some access to water, although the H-bond energy is likely to be larger in low dielectric environments (10). Another relevant model study in tetrahydrofuran has detected a strong H-bond between 3,4-dinitrophenol and 3,4-dinitrophenolate (6.0 kcal/mol) and 4-nitrophenol and 4-nitrophenolate (6.3 kcal/mol) (ApKa = 0) (24).…”

mentioning

confidence: 99%

NMR evidence for the participation of a low-barrier hydrogen bond in the mechanism of delta 5-3-ketosteroid isomerase.

Zhao

Abeygunawardana

Talalay³

et al. 1996

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

131

135

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A5-3-Ketosteroid isomerase (EC 5.3.3.1) promotes an allylic rearrangement involving intramolecular proton transfer via a dienolic intermediate. This enzyme enhances the catalytic rate by a factor of 1010. Two residues, Tyr-14, the general acid that polarizes the steroid 3-carbonyl group and facilitates enolization, and Asp-38 the general base that abstracts and transfers the 4(3-proton to the 6j3-position, contribute 104.7 and 105-6 to the rate increase, respectively. A major mechanistic enigma is the huge disparity between the pK, values of the catalytic groups and their targets. Upon binding of an analog of the dienolate intermediate to isomerase, proton NMR detects a highly deshielded resonance at 18.15 ppm in proximity to aromatic protons, and with a 3-fold preference for protium over deuterium (fractionation factor, 4) = 0.34), consistent with formation of a short, strong (low-barrier) hydrogen bond to Tyr-14. The strength of this hydrogen bond is estimated to be at least 7.1 kcal/mol. This bond is relatively inaccessible to bulk solvent and is pH insensitive. Low-barrier hydrogen bonding of Tyr-14 to the

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Enzymic and Nonenzymic Polarizations of .alpha.,.beta.-Unsaturated Ketosteroids and Phenolic Steroids. Implications for the Roles of Hydrogen Bonding in the Catalytic Mechanism of .DELTA.5-3-Ketosteroid Isomerase

Cited by 27 publications

References 27 publications

The Conserved Serine-Tyrosine Dipeptide in Leishmania donovani Hypoxanthine-guanine Phosphoribosyltransferase Is Essential for Catalytic Activity

The Conserved Serine-Tyrosine Dipeptide in Leishmania donovani Hypoxanthine-guanine Phosphoribosyltransferase Is Essential for Catalytic Activity

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

NMR evidence for the participation of a low-barrier hydrogen bond in the mechanism of delta 5-3-ketosteroid isomerase.

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