Acyl group transfer by proteases forming an acylenzyme intermediate: Kinetic model analysis (including hydrolysis of acylenzyme-nucleophile complex)

Gololobov, Mikhail Yu.; Borisov, Ivan L.; Švedas, Vytas K.

doi:10.1016/s0022-5193(89)80128-6

Cited by 25 publications

(12 citation statements)

References 7 publications

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“…However, it remains possible that this activity exists in one of the many uncharacterized clades of this protein family (see below). The likelihood of this possibility could be tested by engineering a lipoamidase from an amidotransferase, which would be essentially the reverse of peptide transfer by proteases (24).…”

Section: Extractmentioning

confidence: 99%

A Complex Lipoate Utilization Pathway in Listeria monocytogenes

Christensen

Hagar

O’Riordan

et al. 2011

Journal of Biological Chemistry

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Although a complete pathway of lipoic acid metabolism has been established in Escherichia coli, lipoic acid metabolism in other bacteria is more complex and incompletely understood. Listeria monocytogenes has been shown to utilize two lipoateprotein ligases for lipoic acid scavenging, whereas only one of the ligases can function in utilization of host-derived lipoic acidmodified peptides. We report that lipoic acid scavenging requires not only ligation of lipoic acid but also a lipoyl relay pathway in which an amidotransferase transfers lipoyl groups to the enzyme complexes that require the cofactor for activity. In addition, we provide evidence for a new lipoamidase activity that could allow utilization of lipoyl peptides by lipoate-protein ligase. These data support a model of an expanded, three-enzyme pathway for lipoic acid scavenging that seems widespread in the Firmicutes phylum of bacteria.Listeria monocytogenes is a Gram-positive bacterium of the phylum Firmicutes that is a serious human pathogen. Like a number of other bacteria, it is a natural lipoic acid auxotroph and relies upon exogenous sources of lipoic acid for growth. Utilization of lipoic acid requires that the cofactor become covalently attached to the ⑀-amino group of a conserved lysine residue of the lipoyl domain(s) of the enzyme complexes that require the cofactor for activity. The only known route for attachment of exogenous lipoic acid is by lipoic acid ligase action (1). L. monocytogenes employs two lipoate protein ligases for this purpose, LplA1 and LplA2 (2). Although either ligase suffices for lipoylation in cells grown in a rich medium, only lplA1 is essential for intracellular growth and virulence (2, 3). The largest lipoyl peptide substrate utilized by L. monocytogenes is the DKA tripeptide, where lipoate is attached to the lysine ⑀-amino group (DK L A) by an amide linkage. LplA1 is required for efficient use of DK L A as a lipoate source (2). Prior work (2) indicated that expression of LplA1 but not LplA2 in Escherichia coli functionally replaced the host LplA ligase, the most thoroughly characterized lipoate ligase. The enzymatic properties that differentiate LplA1 from LplA2 were unknown.It was proposed that LplA1 and LplA2 may have different protein interaction partners or that LplA1 may be able to transfer the lipoyl group from a synthetic lipoylated peptide, DK L A, to lipoate requiring enzymes by functioning as a lipoyl-amidotransferase (2). We conducted the present study to improve our understanding of lipoyl scavenge by L. monocytogenes and to determine the mechanism of DK L A utilization. It was possible that the DK L A lipoyl moiety could be utilized either by direct transfer of the lipoyl moiety or by hydrolysis with subsequent ligation of the lipoic acid released.In Bacillus subtilis a novel amidotransferase, called LipL, was recently shown to be required for lipoic acid biosynthesis (4, 5). Moreover, LipL homologues are present in all Firmicutes that use lipoic acid including those, such as L. monocytogenes, that lac...

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Section: Extractmentioning

confidence: 99%

A Complex Lipoate Utilization Pathway in Listeria monocytogenes

Christensen

Hagar

O’Riordan

et al. 2011

Journal of Biological Chemistry

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“…1. Kinetic scheme describing the synthesis and hydrolysis reactions catalysed by penicillin acylase, adapted from a similar scheme for chymotrypsin catalysed reactions [7]. In this Scheme A(D) is the acyl donor, N the b-lactam nucleophile, Q the product of aminolysis (i.e.…”

Section: Kinetic Measurementsmentioning

confidence: 99%

“…For the deacylation such information is more difficult to obtain since the individual rate and binding constants cannot be measured separately. Some information may be obtained by performing nucleophile competition experiments [7]. The rate of aminolysis vs. hydrolysis can be expressed as the V s /V h ratio and according to Scheme 1, is given by [33]:…”

Section: Deacylation By B-lactam Nucleophilesmentioning

confidence: 99%

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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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“…Papers [81, 82] contain a detailed analysis of this scheme and its various derivatives. The basic result following from this analysis is that the description of the process’s efficiency cannot be accomplished by using such common parameters as catalytic constants, Michaelis constants, or constants of inhibition by the reaction products.…”

Section: Pa-g Protein Engineeringmentioning

confidence: 99%