Electrophilic catalysis can explain the unexpected acidity of carbon acids in enzyme-catalyzed reactions

Gerlt, J.A.; Kozarich, John W.; Kenyon, George L.; Gassman, Paul G.

doi:10.1021/ja00025a039

Cited by 166 publications

(162 citation statements)

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“…It is proposed that an electrophilic catalyst positioned adjacent to the carbonyl oxygen of the substrate is primarily responsible for stabilizing the intermediates (10). The available three-dimensional structure of KSI complexed with equilenin confirms that, invariably, electrophilic catalysts are positioned proximal to the carbonyl group of the substrate at the active site as suggested previously (11).…”

Section: Discussionsupporting

confidence: 82%

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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Section: Discussionsupporting

confidence: 82%

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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“…To explain the high catalytic rate, considerable substrate activation, which effectively decreases the pK a of the R-proton and is equivalent to the stabilization of an enolate-like intermediate species, has to occur. In the past, similar arguments have been made for analogous enzyme systems that are involved in the deprotonation of R-carbon acid substrates (10).…”

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confidence: 69%

Substrate Activation by Acyl-CoA Dehydrogenases: Transition-State Stabilization and pKs of Involved Functional Groups

et al. 1998

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ABSTRACT:The mechanism by which acyl-CoA dehydrogenases initiate catalysis was studied by using p-substituted phenylacetyl-CoAs (substituents -NO 2 , -CN, and CH 3 CO-), 3S-C 8 -, and 3′-dephospho-3S-C 8 CoA. These analogues lack a C-H and cannot undergo R, -dehydrogenation. Instead they deprotonate at RC-H at pH g 14 to form delocalized carbanions having strong absorbancies in the near UV-visible spectrum. The pK a s of the corresponding phenylacetone analogues were determined as ≈13.6 (-NO 2 ), ≈14.5 (-CN), and ≈14.6 (CH 3 CO-). Upon binding to human wild-type medium-chain acyl-CoA dehydrogenase (MCADH), all analogues undergo RC-H deprotonation. While the extent of deprotonation varies, the anionic products form charge-transfer complexes with the oxidized flavin. From the pH dependence of the dissociation constants (K d ) of p-NO 2 -phenylacetyl-CoA (4NPA-CoA), 3S-C 8 -CoA, and 3′-dephospho-3S-C 8 CoA, four pK a s at ≈5, ≈6, ≈7.3, and ≈8 were identified. They were assigned to the following ionizations: (a) pK a ≈5, ligand (L-H) in the MCADH∼ligand complex; (b) pK a ≈6, Glu376-COOH in uncomplexed MCADH; (c) pK a ≈7.3, Glu99-COOH in uncomplexed MCADH (Glu99 is a residue that flanks the bottom of the active-center cavity; this pK is absent in the mutant Glu99Gly-MCADH); and (d) pK ≈8, Glu99-COOH in the MCADH∼4NPA-CoA complex. The pK a ≈6 (b) is not significantly affected in the MCADH∼4NPA-CoA complex, but it is increased by g1 pK unit in that with 3S-C 8 CoA and further in the presence of C 8 -CoA, the best substrate. The RC-H pK a s of 4NPA-CoA, of 3S-C 8 -CoA, and of 3′-dephospho-3S-C 8 CoA in the complex with MCADH are ≈5, ≈5, and ≈6. Compared to those of the free species these pK a values are therefore lowered by 8 to g11 pH units (50 to g 65 kJ mol -1 ) and are close to the pK a of Glu376-COOH in the complex with substrate/ligand. This effect is ascribed mainly to the hydrogen-bond interactions of the thioester carbonyl group with the ribityl-2′-OH of FAD and Glu376-NH. It is concluded that the pK a shifts induced with normal substrates such as n-octanoyl-CoA are still higher and of the order of 9-13 pK units. With 4NPA-CoA and MCADH, RC-H abstraction is fast (k app ≈55 s -1 at pH 7.5 and 25°C, deuterium isotope effect ≈1.34). However, it does not proceed to completion since it constitutes an approach to equilibrium with a finite rate for reprotonation in the pH range 6-9.5. The extent of deprotonation and the respective rates are pH-dependent and reflect apparent pK a s of ≈5 and ≈7.3, which correspond to those determined in static experiments.Acyl-CoA dehydrogenases catalyze the R, -dehydrogenation of fatty acid acyl-CoA conjugates to their corresponding enoyl-CoA products; the redox equivalents formed in this reaction are transferred to electron transferring flavoprotein and further to the respiratory chain (1, 2). A peculiarity of the R, -dehydrogenation reaction is that it involves the concomitant fission of two kinetically stable C-H bonds. In the past, studies with medium-chain acyl-CoA dehydrogenase ...

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“…In the R218K-(Ca 2ϩ ) 3-4 -pentaGalpA structures, the carboxyl group of GalpA 3 is neutralized by interactions with 3 Ca 2ϩ and 2 Ca 2ϩ as well as by , an invariant amino acid in the pectate lyase subfamily. Lys-190 also may serve an additional role, which is to partially protonate the carboxylic acid group, stabilizing an enolic intermediate as postulated and defined by Gerlt and colleagues (Gerlt et al, 1991;Gassman, 1992, 1993). Either or both effects serve to decrease the pK a (the negative log of the dissociation constant) of the ␣ proton at C-5, making it more susceptible to an attack by a base.…”

Section: Discussionmentioning

confidence: 92%

Structure of a Plant Cell Wall Fragment Complexed to Pectate Lyase C

et al. 1999

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The three-dimensional structure of a complex between the pectate lyase C (PelC) R218K mutant and a plant cell wall fragment has been determined by x-ray diffraction techniques to a resolution of 2.2 Å and refined to a crystallographic R factor of 18. Because the R218K PelC-galacturonopentaose complex represents an intermediate in the reaction pathway, the structure also reveals important details regarding the enzymatic mechanism. Notably, the results suggest that an arginine, which is invariant in the pectate lyase superfamily, is the amino acid that initiates proton abstraction during the ␤ elimination cleavage of polygalacturonic acid. INTRODUCTIONPectate lyases are depolymerizing enzymes that degrade plant cell walls, causing tissue maceration and death. The enzymes normally are secreted by phytopathogenic organisms and are known to be the primary virulence agents in soft rot diseases caused by Erwinia spp (Collmer and Keen, 1986;Kotoujansky, 1987;Barras et al., 1994). In the latter organisms, the enzymes exist as multiple, independently regulated isozymes that share amino acid sequence identity ranging from 27 to 80%.Pectate lyases share sequence similarities with fungal pectin lyases, plant pollen proteins, and plant style proteins (Henrissat et al., 1996). The three-dimensional structures of five members of the superfamily have been determined and include Erwinia chrysanthemi pectate lyase C (PelC) (Yoder et al., 1993;Yoder and Jurnak, 1995), E. chrysanthemi pectate lyase E (PelE) (Lietzke et al., 1994), Bacillus subtilis pectate lyase ( B. subtilis Pel) (Pickersgill et al., 1994), Aspergillus niger pectin lyase A (PLA) (Mayans et al., 1997), and A. niger pectin lyase B (PLB) (Vitali et al., 1998). All share a similar but an unusual structural motif, termed the parallel ␤ helix, in which the ␤ strands are folded into a large, right-handed coil. The enzyme structures differ in the size and conformation of the loops that protrude from the parallel ␤ helix core. As deduced from sequence similarity and site-directed mutagenesis studies, the protruding loops on one side of the parallel ␤ helix form the pectolytic active site (Kita et al., 1996). The structural differences of the loops are believed to be related to subtle differences in the enzymatic and maceration properties of the proteins.Pectate lyases catalyze the cleavage of pectate, the deesterified product of pectin, which is the major component that maintains the structural integrity of cell walls in higher plants The Plant Cell (Carpita and Gibeaut, 1993). The pectate backbone is composed of blocks of polygalacturonic acid (PGA), which is a helical homopolymer of D -galacturonic acid (Gal p A) units linked by ␣ -(1 → 4) glycosidic bonds. The blocks of PGA are separated by stretches in which (1 → 2)-␣ -L -rhamnose residues alternate with Gal p A (McNaught, 1997). Blocks of PGA may contain as many as 200 Gal p A units and span 100 nm (Thibault et al., 1993). Cations are necessary to neutralize PGA in solution and, as a consequence, influence its struct...

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Electrophilic catalysis can explain the unexpected acidity of carbon acids in enzyme-catalyzed reactions

Cited by 166 publications

References 0 publications

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

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

Substrate Activation by Acyl-CoA Dehydrogenases: Transition-State Stabilization and pKs of Involved Functional Groups

Structure of a Plant Cell Wall Fragment Complexed to Pectate Lyase C

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