KPC-2 β-lactamase Enables Carbapenem Antibiotic Resistance Through Fast Deacylation of the Covalent Intermediate

Serine active-site β-lactamases hydrolyze β-lactam antibiotics through formation of a covalent acyl-enzyme intermediate followed by deacylation via an activated water molecule. Carbapenem antibiotics are poorly hydrolyzed by most β-lactamases due to slow hydrolysis of the acyl-enzyme intermediate. However, the emergence of the KPC-2 carbapenemase has resulted in widespread resistance to these drugs, suggesting it operates more efficiently. Here, we investigated the unusual features of KPC-2 that enable this resistance. We show that KPC-2 has a 20,000-fold increased deacylation rate compared to the common TEM-1 β-lactamase. Further, kinetic analysis of active site alanine mutants indicates that carbapenem hydrolysis is a concerted effort involving multiple residues. Substitution of Asn170 greatly decreases the deacylation rate, but this residue is conserved in both KPC-2 and non-carbapenemase β-lactamases, suggesting it promotes carbapenem hydrolysis only in the context of KPC-2. X-ray structure determination of the N170A enzyme in complex with hydrolyzed imipenem suggests Asn170 may prevent the inactivation of the deacylating water by the 6α-hydroxyethyl substituent of carbapenems. In addition, the Thr235 residue, which interacts with the C3 carboxylate of carbapenems, also contributes strongly to the deacylation reaction. In contrast, mutation of the Arg220 and Thr237 residues decreases the acylation rate and, paradoxically, improves binding affinity for carbapenems. Thus, the role of these residues may be ground state destabilization of the enzyme-substrate complex or, alternatively, to ensure proper alignment of the substrate with key catalytic residues to facilitate acylation. These findings suggest modifications of the carbapenem scaffold to avoid hydrolysis by KPC-2 β-lactamase.

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Effect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B

Canadian Journal of Biochemistry ◽

10.1139/o75-102 ◽

1975 ◽

Vol 53 (7) ◽

pp. 747-757 ◽

Cited By ~ 3

Author(s):

Graham J. Moore ◽

N. Leo Benoiton

Keyword(s):

Active Site ◽

Active Sites ◽

Kinetic Behavior ◽

Carboxypeptidase A ◽

Phenyllactic Acid ◽

Carboxypeptidase B ◽

Substrate Complex ◽

Enzyme Substrate ◽

Basic Peptides ◽

Hydrolysis Of

The initial rates of hydrolysis of Bz-Gly-Lys and Bz-Gly-Phe by carboxypeptidase B (CPB) are increased in the presence of the modifiers β-phenylpropionic acid, cyclohexanol, Bz-Gly, and Bz-Gly-Gly. The hydrolysis of the tripeptide Bz-Gly-Gly-Phe is also activated by Bz-Gly and Bz-Gly-Gly, but none of these modifiers activate the hydrolysis of Bz-Gly-Gly-Lys, Z-Leu-Ala-Phe, or Bz-Gly-phenyllactic acid by CPB. All modifiers except cyclohexanol display inhibitory modes of binding when present in high concentration.Examination of Lineweaver–Burk plots in the presence of fixed concentrations of Bz-Gly has shown that activation of the hydrolysis of neutral and basic peptides by CPB, as reflected in the values of the extrapolated parameters, Km(app) and keat, occurs by different mechanisms. For Bz-Gly-Gly-Phe, activation occurs because the enzyme–modifier complex has a higher affinity than the free enzyme for the substrate, whereas activation of the hydrolysis of Bz-Gly-Lys derives from an increase in the rate of breakdown of the enzyme–substrate complex to give products.Cyclohexanol differs from Bz-Gly and Bz-Gly-Gly in that it displays no inhibitory mode of binding with any of the substrates examined, activates only the hydrolysis of dipeptides by CPB, and has a greater effect on the hydrolysis of the basic dipeptide than on the neutral dipeptide. Moreover, when Bz-Gly-Lys is the substrate, cyclohexanol activates its hydrolysis by CPB by increasing both the enzyme–substrate binding affinity and the rate of the catalytic step, an effect different from that observed when Bz-Gly is the modifier.The anomalous kinetic behavior of CPB is remarkably similar to that of carboxypeptidase A, and is a good indication that both enzymes have very similar structures in and around their respective active sites. A binding site for activator molecules down the cleft of the active site is proposed for CPB to explain the observed kinetic behavior.

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Human cholinesterases

ORGANOPHOSPHORUS NEUROTOXINS ◽

10.29039/chapter_5e4132b5f22366.15634219 ◽

2020 ◽

pp. 63-120

Author(s):

Sergey Varfolomeev ◽

Bella Grigorenko ◽

Sofya Lushchekina ◽

Alexander Nemuchin

Keyword(s):

Active Site ◽

The Body ◽

Polymorphic Modification ◽

Substrate Complex ◽

Enzyme Substrate ◽

Mechanism Of Hydrolysis ◽

The Central Nervous System ◽

The Stability ◽

Hydrolysis Of ◽

Enzyme Reactivity

The work is devoted to modeling the elementary stages of the hydrolysis reaction in the active site of enzymes belonging to the class of cholinesterases — acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The study allowed to describe at the molecular level the effect of the polymorphic modification of BChE, causing serious physiolog ical consequences. Cholinesterase plays a crucial role in the human body. AChE is one of the key enzymes of the central nervous system, and BChE performs protective functions in the body. According to the results of calculations using the combined method of quantum and molecular mechanics (KM/MM), the mechanism of the hydrolysis of the native acetylcholine substrate in the AChE active center was detailed. For a series of ester substrates, a method for estimation of dependence of the enzyme reactivity on the structure of the substrate has been developed. The mechanism of hydrolysis of the muscle relaxant of succininylcholine BChE and the effect of the Asp70Gly polymorph on it were studied. Using various computer simulation methods, the stability of the enzyme-substrate complex of two enzyme variants with succinylcholine was studied.

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Role of Sulfated Tyrosines of Thyroglobulin in Thyroid Hormonosynthesis

Endocrinology ◽

10.1210/en.2005-0197 ◽

2005 ◽

Vol 146 (11) ◽

pp. 4834-4843 ◽

Cited By ~ 4

Author(s):

Marie-Christine Nlend ◽

David M. Cauvi ◽

Nicole Venot ◽

Odile Chabaud

Keyword(s):

Amino Acid ◽

Coupling Reaction ◽

Short Life ◽

Hormone Synthesis ◽

Amino Acid Peptide ◽

Substrate Complex ◽

Enzyme Substrate ◽

Saturation Binding ◽

Hydrolysis Of

Our previous studies showed that sulfated tyrosines (Tyr-S) are involved in thyroid hormone synthesis and that Tyr5, the main hormonogenic site of thyroglobulin (Tg), is sulfated. In the present paper, we studied the role of Tyr-S in the formation and activity of the peroxidase-Tg complex. Results show that noniodinated 35SO3-Tg specifically binds (Kd = 1.758 μm) to immobilized lactoperoxidase (LPO) via Tyr-S linkage by using saturation binding and competition experiments. We found that NIFEY-S, a 15-amino acid peptide corresponding to the NH2-end sequence of Tg and containing the hormonogenic acceptor Tyr5-S, was a better competitor than cholecystokinin and Tyr-S. 35SO3-Tg, iodinated without peroxidase, bound to LPO with a Kd (1.668 μm) similar to that of noniodinated Tg, suggesting that 1) its binding occurs via Tyr-S linkage and 2) Tyr-S requires peroxidase to be iodinated, whereas nonsulfated Tyr does not. Iodination of NIFEY-S with [125I]iodide showed that Tyr5-S iodination increased with LPO concentration, whereas iodination of a nonsulfated peptide containing the donor Tyr130 was barely dependent on LPO concentration. Enzymatic hydrolysis of iodinated Tg or NIFEY-S showed that the amounts of sulfated iodotyrosines also depended on LPO amount. Sulfated iodotyrosines were detectable in the enzyme-substrate complex, suggesting they have a short life before the coupling reaction occurs. Our data suggest that after Tyr-S binding to peroxidase where it is iodinated, the sulfate group is removed, releasing an iodophenoxy anion available for coupling with an iodotyrosine donor.

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The Role of Evolving Interfacial Substrate Properties on Heterogeneous Cellulose Hydrolysis Kinetics

10.1101/691071 ◽

2019 ◽

Cited By ~ 1

Author(s):

Jennifer Nill ◽

Tina Jeoh

Keyword(s):

Binding Sites ◽

Binding Capacity ◽

Cellulose Hydrolysis ◽

Reaction Rates ◽

Hydrolysis Kinetics ◽

Substrate Complex ◽

Enzyme Substrate ◽

Substrate Properties ◽

Hydrolysis Of

AbstractInterfacial enzyme reactions require formation of an enzyme-substrate complex at the surface of a heterogeneous substrate, but often multiple modes of enzyme binding and types of binding sites complicate analysis of their kinetics. Excess of heterogeneous substrate is often used as a justification to model the substrate as unchanging; but using the study of the enzymatic hydrolysis of insoluble cellulose as an example, we argue that reaction rates are dependent on evolving substrate interfacial properties. We hypothesize that the relative abundance of binding sites on cellulose where hydrolysis can occur (productive binding sites) and binding sites where hydrolysis cannot be initiated or is inhibited (non-productive binding sites) contribute to rate limitations. We show that the initial total number of productive binding sites (the productive binding capacity) determines the magnitude of the initial burst phase of cellulose hydrolysis, while productive binding site depletion explains overall hydrolysis kinetics. Furthermore, we show that irreversibly bound surface enzymes contribute to the depletion of productive binding sites. Our model shows that increasing the ratio of productive- to non-productive binding sites promotes hydrolysis, while maintaining an elevated productive binding capacity throughout conversion is key to preventing hydrolysis slowdown.

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Peptide substrates for chymosin (rennin). Interaction sites in κ-casein-related sequences located outside the (103-108)-hexapeptide region that fits into the enzyme's active-site cleft

Biochemical Journal ◽

10.1042/bj2440553 ◽

1987 ◽

Vol 244 (3) ◽

pp. 553-558 ◽

Cited By ~ 42

Author(s):

S Visser ◽

C J Slangen ◽

P J van Rooijen

Keyword(s):

Active Site ◽

Peptide Bond ◽

Milk Clotting ◽

Substrate Complex ◽

Interaction Sites ◽

Enzyme Substrate ◽

Ability To Act ◽

Milk Clotting Enzyme ◽

Active Site Cleft

The role of individual amino acid residues in the 98-102 and 111-112 regions of bovine kappa-casein in its interaction with the milk-clotting enzyme chymosin (rennin) was investigated. to this end the tryptic 98-112 fragment of kappa-casein was modified in its N- and/or C-terminal part by chemical (guanidation, ethoxyformylation, repeated Edman degradation) and enzymic (carboxypeptidase) treatments. Further, use was made of short synthetic kappa-casein analogues in which His-102 had been replaced by Pro or Lys. All peptides and their derivatives were tested comparatively at various pH values for their ability to act as chymosin substrates via specific cleavage of the peptide bond at position 105-106. The results indicate that in the alternating 98-102 sequence (His-Pro-His-Pro-His) the His as well as the Pro residues contribute to the substrate activity with no predominant role of any one of these groups. Another interaction site is formed by the Lys residue at position 111 of the substrate. A model of the enzyme-substrate complex is proposed. Herein the 103-108 fragment of the substrate, to be accommodated within the enzyme's active-site cleft, is brought into position by electrostatic binding (via His-98, His-100, His-102 and Lys-111) near the entrance of the cleft. These interactions are strongly supported by Pro residues at positions 99, 101, 109 and 110 of the substrate, which act as stabilizers of the proper conformation of the substrate in the enzyme-substrate complex.

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Human cholinesterases

ORGANOPHOSPHORUS NEUROTOXINS ◽

10.29039/21_069-126 ◽

2020 ◽

pp. 69-126

Author(s):

Sergey Varfolomeev ◽

Bella Grigorenko ◽

Sofya Lushchekina ◽

Patrick Masson ◽

Galina Mahaeva ◽

...

Keyword(s):

Active Site ◽

The Body ◽

Polymorphic Modification ◽

Substrate Complex ◽

Enzyme Substrate ◽

Mechanism Of Hydrolysis ◽

The Central Nervous System ◽

The Stability ◽

Hydrolysis Of ◽

Enzyme Reactivity

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Mechanistic consequences of replacing the active-site nucleophile Glu-358 in Agrobacterium sp. β-glucosidase with a cysteine residue

Biochemical Journal ◽

10.1042/bj3300203 ◽

1998 ◽

Vol 330 (1) ◽

pp. 203-209 ◽

Cited By ~ 12

Author(s):

L. Sherry LAWSON ◽

J. R. Antony WARREN ◽

G. Stephen WITHERS

Keyword(s):

Rate Constant ◽

Active Site ◽

Cysteine Residue ◽

Kinetic Evaluation ◽

Glycosidic Bonds ◽

Proposal A ◽

Leaving Group ◽

Hydrolysis Of ◽

Enzyme Intermediate

Retaining glycosidases achieve the hydrolysis of glycosidic bonds through the assistance of two key active-site carboxyls. One carboxyl functions as a nucleophile/leaving group, and the other acts as the acid-base catalyst. It has been suggested that a cysteine residue could fulfil the role of the active site nucleophile [Hardy and Poteete (1991) Biochemistry 30, 9457-9463]. To test the validity of this proposal, a kinetic evaluation was conducted on the active-site nucleophile cysteine mutant (Glu-358 → Cys) of the retaining β-glucosidase from Agrobacterium sp. The Glu-358 → Cys mutant was able to complete the first step (glycosylation) of the enzymic mechanism, forming a covalent glycosyl-enzyme intermediate, but the rate constant for this step was decreased to 1/106 of that of the native enzyme. The subsequent hydrolysis (deglycosylation) step was also severely affected by the replacement of Glu-358 with a cysteine residue, with the rate constant being depressed to 1/107 or less. Thus Cys-358 functions inefficiently in both the capacity of catalytic nucleophile and leaving group. On the basis of these results it seems unlikely that the role of the active-site nucleophile in retaining glycosidases could successfully be filled by a cysteine residue.

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Structural studies of duck δ2 crystallin mutants provide insight into the role of Thr161 and the 280s loop in catalysis

Biochemical Journal ◽

10.1042/bj20040656 ◽

2004 ◽

Vol 384 (2) ◽

pp. 437-447 ◽

Cited By ~ 11

Author(s):

Liliana M. SAMPALEANU ◽

Penelope W. CODDING ◽

Yuri D. LOBSANOV ◽

May TSAI ◽

G. David SMITH ◽

...

Keyword(s):

Active Site ◽

Side Chain ◽

Structural Studies ◽

Cycle Enzyme ◽

Substrate Complex ◽

Enzyme Substrate ◽

Catalytically Active ◽

Urea Cycle Enzyme ◽

Positively Charged

δ Crystallin, a taxon-specific crystallin present in avian eye lenses, is homologous to the urea cycle enzyme ASL (argininosuccinate lyase). Although there are two δ crystallin isoforms in duck lenses, dδc1 (duck δ1 crystallin) and dδc2 (duck δ2 crystallin), only dδc2 is catalytically active. Previous structural studies have suggested that residues Ser283 and His162 in the multi-subunit active site of dδc2/ASL are the putative catalytic acid/base, while the highly conserved, positively charged Lys289 is thought to help stabilize the carbanion intermediate. The strict conservation of a small hydroxy-containing residue (Thr or Ser) at position 161 adjacent to the putative catalytic base, as well as its proximity to the substrate in the S283A dδc2 enzyme–substrate complex, prompted us to investigate further the role this residue. Structures of the active T161S and inactive T161D dδc2 mutants, as well as T161D complexed with argininosuccinate, have been determined to 2.0 Å resolution. The structures suggest that a hydroxy group is required at position 161 to help correctly position the side chain of Lys289 and the fumarate moiety of the substrate. Threonine is probably favoured over serine, because the interaction of its methyl group with Leu206 would restrict its conformational flexibility. Residues larger than Thr or Ser interfere with substrate binding, supporting previous suggestions that correct positioning of the substrate's fumarate moiety is essential for catalysis to occur. The presence of the 280s loop (i.e. a loop formed by residues 270–290) in the ‘open’ conformation suggests that loop closure, thought to be essential for sequestration of the substrate, may be triggered by the formation of the carbanion or aci-carboxylate intermediates, whose charge distribution more closely mimics that of the sulphate ion found in the active-site region of the inactive dδc1. The 280s loop in dδc1 is in the closed conformation.

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The role of secondary alcoholic groups of D-galacturonan in its degradation with exo-D-galacturonanase

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19800427 ◽

1980 ◽

Vol 45 (2) ◽

pp. 427-434 ◽

Cited By ~ 4

Author(s):

Kveta Heinrichová ◽

Rudolf Kohn

Keyword(s):

Acetyl Derivative ◽

Competitive Inhibitor ◽

Hydroxyl Groups ◽

Pectic Acid ◽

Substrate Complex ◽

Enzyme Substrate ◽

The Individual ◽

Degradation Degree ◽

Derivatives Of

The effect of exo-D-galacturonanase from carrot on O-acetyl derivatives of pectic acid of variousacetylation degree was studied. Substitution of hydroxyl groups at C(2) and C(3) of D-galactopyranuronic acid units influences the initial rate of degradation, degree of degradation and its maximum rate, the differences being found also in the time of limit degradations of the individual O-acetyl derivatives. Value of the apparent Michaelis constant increases with increase of substitution and value of Vmax changes. O-Acetyl derivatives act as a competitive inhibitor of degradation of D-galacturonan. The extent of the inhibition effect depends on the degree of substitution. The only product of enzymic reaction is D-galactopyranuronic acid, what indicates that no degradation of the terminal substituted unit of O-acetyl derivative of pectic acid takes place. Substitution of hydroxyl groups influences the affinity of the enzyme towards the modified substrate. The results let us presume that hydroxyl groups at C(2) and C(3) of galacturonic unit of pectic acid are essential for formation of the enzyme-substrate complex.

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Kinetics and Mechanism of Hydrolysis of Acetylthiocholine by Butyrylcholine Esterase

Zeitschrift für Naturforschung C ◽

10.1515/znc-2002-11-1221 ◽

2002 ◽

Vol 57 (11-12) ◽

pp. 1072-1077 ◽

Cited By ~ 4

Author(s):

Karel Komers ◽

Alexandr Čegan ◽

Marek Link

Keyword(s):

Acetic Acid ◽

Kinetics And Mechanism ◽

Kinetics Parameters ◽

Mechanism Of Hydrolysis ◽

Ph Stat ◽

Stat Method ◽

Ellman’S Method ◽

Hydrolysis Of

Kinetics and mechanism of hydrolysis of acetylthiocholine by the enzyme butyrylcholine esterase was studied. The spectrophotometric Ellman’s method and potentiometric pH-stat method were used for continuous determination of the actual concentration of the products thiocholine and acetic acid in the reaction mixture. The validity of the Michaelis-Menten (Briggs-Haldane) equation in the whole course of the reaction under used conditions was proved. The corresponding kinetics parameters (Vm and KM) were calculated from the obtained dependences of concentration of thiocholine or acetic acid vs. time and compared. From this comparison the deciding kinetic role of the step producing thiocholine was derived. The values of initial molar concentration of the enzyme and of the rate constants of the kinetic model were estimated.

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