Correlative variations in enzyme-derived and substrate-derived structures of catalytic transition states. Implications for the catalytic strategy of acyl-transfer enzymes

Stein, Ross L.; Elrod, James P.; Schowen, Richard L.

doi:10.1021/ja00346a056

Cited by 43 publications

(38 citation statements)

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“…The results of the proton inventories indicate that the mechanisms for the conversion of the substrates that were studied, differ significantly from each other. Substrate-dependent proton inventories have also been observed for the serine proteases elastase [31] and chymotrypsin [32]. The results of the proton inventories for NIPAB and NIPAOB suggest that also in PA-catalysed reactions the structure of the leaving group of the substrate has an effect on the details of the kinetic mechanism.…”

Section: Proton Transfer In the Acylation Reactionmentioning

confidence: 90%

Kinetics of enzyme acylation and deacylation in the penicillin acylase‐catalyzed synthesis of β‐lactam antibiotics

Alkema

Vries

Floris

et al. 2003

European Journal of Biochemistry

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Penicillin acylase catalyses the hydrolysis and synthesis of semisynthetic b-lactam antibiotics via formation of a covalent acyl-enzyme intermediate. The kinetic and mechanistic aspects of these reactions were studied. Stopped-flow experiments with the penicillin and ampicillin analogues 2-nitro-5-phenylacetoxy-benzoic acid (NIPAOB) and D-2-nitro-5-[(phenylglycyl)amino]-benzoic acid (NIPGB) showed that the rate-limiting step in the conversion of penicillin G and ampicillin is the formation of the acylenzyme. The phenylacetyl-and phenylglycyl-enzymes are hydrolysed with rate constants of at least 1000 s )1 and 75 s )1 , respectively. A normal solvent deuterium kinetic isotope effect (KIE) of 2 on the hydrolysis of 2-nitro-5-[(phenylacetyl)amino]-benzoic acid (NIPAB), NIPGB and NIPAOB indicated that the formation of the acyl-enzyme proceeds via a general acid-base mechanism. In agreement with such a mechanism, the proton inventory of the k cat for NIPAB showed that one proton, with a fractionation factor of 0.5, is transferred in the transition state of the rate-limiting step. The overall KIE of 2 for the k cat of NIPAOB resulted from an inverse isotope effect at low concentrations of D 2 O, which is overridden by a large normal isotope effect at large molar fractions of D 2 O. Rate measurements in the presence of glycerol indicated that the inverse isotope effect originated from the higher viscosity of D 2 O compared to H 2 O. Deacylation of the acyl-enzyme was studied by nucleophile competition and inhibition experiments. The b-lactam compound 7-aminodesacetoxycephalosporanic acid (7-ADCA) was a better nucleophile than 6-aminopenicillanic acid, caused by a higher affinity of the enzyme for 7-ADCA and complete suppression of hydrolysis of the acyl-enzyme upon binding of 7-ADCA. By combining the results of the steadystate, presteady state and nucleophile binding experiments, values for the relevant kinetic constants for the synthesis and hydrolysis of b-lactam antibiotics were obtained.Keywords: penicillin acylase; antibiotic synthesis; kinetic mechanism; kinetic isotope effect.Penicillin acylase (PA) (EC 3.5.1.11) of Escherichia coli ATCC 11105 hydrolyses penicillin G to produce phenylacetic acid and 6-aminopenicillanic acid (6-APA). The latter compound can be used industrially as a precursor for the synthesis of semisynthetic b-lactam antibiotics. In synthetic reactions, 6-APA is coupled to an acyl group in a kinetically controlled conversion in which the acyl group is supplied as an activated precursor, e.g. an ester or an amide [1]. This condensation reaction can also be catalysed by PA and the yield of the desired product is strongly dependent on the mechanism and kinetic properties of the catalyst [2].The catalytic mechanism of PA involves nucleophilic attack of the active-site serine, bS1, on the carbonyl carbon of the amide or ester bond of the substrate [3] (residues are labelled to indicate the subunit (a or b) and their position in the subunit). Via a tetrahedral intermediate that is stabilized by hyd...

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Section: Proton Transfer In the Acylation Reactionmentioning

confidence: 90%

Kinetics of enzyme acylation and deacylation in the penicillin acylase‐catalyzed synthesis of β‐lactam antibiotics

Alkema

Vries

Floris

et al. 2003

European Journal of Biochemistry

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“…When the tetrahedral intermediate is broken in the direction of products, the pK a of Asp"!# can return to its original value, owing to the reorganization of the hydrogen-bonded network. This model is further supported by the β-#H secondaryisotope effect, the kinetic #H # O solvent-isotope effect and protoninventory results obtained with serine proteases [26,27]. Substrates that lack most of the structural features of a natural substrate and can be considered only little or not at all to affect the enzyme conformation, exhibit one-proton catalysis, whereas more specific substrates result in two-proton (or multiple-proton) catalysis.…”

Section: Enzyme-substrate Interaction In the Catalytic Triad Of Serinmentioning

confidence: 69%

“…This suggests an increase in the pK a of Asp"!# during the reaction. In the following discussion I will show experimental evidence for the change in the pK a of Asp"!# by reinterpreting previous NMR [5,6] and isotope-effect [26,27] studies. pK a adjustment has been previously proposed for ionizing groups of serine proteases [8,16,[28][29][30][31], but direct experimental evidence for a change in the pK a of Asp"!# has not been described.…”

Section: Enzyme-substrate Interaction In the Catalytic Triad Of Serinmentioning

confidence: 91%

Enzyme–substrate interaction in the catalytic triad of serine proteases: increase in the pKa of Asp102

Neuvonen

1997

Biochemical Journal

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“…Unlike the chymotrypsincatalyzed hydrolysis of similar substrates (e.g., Ac-Tyr p-nitrophenyl ester) where acylation chemistry is rapid and k c /K m is rate-limited by the binding of substrate, producing solvent isotope effects of unity, the hydrolysis of Z-Val p-nitrophenyl ester by elastase is rate-limited by chemistry. For such substrates, linear proton inventories are the rule [6,8,[11][12][13][14]. Additional insight into this reaction step was gained from the proton inventory for this reaction which was observed to be linear, suggesting the existence of a single protonic bridge in the transition state.…”

Section: Activation Of Nucleophile -First Step Of Double Displacementmentioning

confidence: 98%

Proton Transfer during Catalysis by Hydrolases

Stein

2006

Hydrogen‐Transfer Reactions

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Correlative variations in enzyme-derived and substrate-derived structures of catalytic transition states. Implications for the catalytic strategy of acyl-transfer enzymes

Cited by 43 publications

References 1 publication

Kinetics of enzyme acylation and deacylation in the penicillin acylase‐catalyzed synthesis of β‐lactam antibiotics

Kinetics of enzyme acylation and deacylation in the penicillin acylase‐catalyzed synthesis of β‐lactam antibiotics

Enzyme–substrate interaction in the catalytic triad of serine proteases: increase in the pKa of Asp102

Proton Transfer during Catalysis by Hydrolases

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