The ficin-catalysed hydrolysis of p
-nitrophenyl hippurate. Detailed kinetics including the measurement of the apparent dissociation constant for the enzyme-substrate complex

Tervalent cations of the lanthanide (rare-earth) elements reversibly inhibit bacterial collagenase (clostridiopeptidase A; EC 3.4.24.3). Sm3+, whose ionic radius is closest to that of Ca2+, is the most effective inhibitor, completely suppressing clostridiopeptidase activity at a concentration of 100μm in the presence of 5mm-Ca2+. Er3+ and Lu3+, which both have ionic radii smaller than either Ca2+ or Sm3+, inhibit less efficiently, and La3+, which is slightly larger than Ca2+ or Sm3+, inhibits only weakly. These findings indicate a closely fitting, stereospecific, Ca2+-binding pocket in clostridiopeptidase, which excludes ions that are only slightly larger than Ca2+ [ionic radius 0.099nm (0.99 Ȧ)]. By contrast, trypsin, an enzyme whose activity does not depend on Ca2+, requires lanthanide concentrations 50–100-fold greater for inhibition. Furthermore, the relative efficiency of inhibition of trypsin by lanthanides increases as the lanthanide ions become smaller and the charge/volume ratio increases. At a concentration of 50μm, Sm3+ lowers the apparent Km for the hydrolysis of Pz-peptide by clostridiopeptidase from 5.4mm to 0.37mm and the apparent Vmax. from 0.29 Wünsch–Heidrich unit to 0.018 unit. Thus Sm3+ enhances the affinity of this enzyme for its substrate; inhibition of hydrolysis of Pz-peptide may result from the excessive stability of the enzyme–Sm3+–substrate complex. Inhibition by Sm3+ is competitive with regard to Ca2+. The apparent dissociation constant, Kd, of Ca2+ is 0.27mm, where the Ki for Sm3+ is 12μm. Clostridiopeptidase is more thermolabile in the absence of Ca2+. With Sm3+, thermoinactivation of the enzyme at 53°C or 60°C is initially accelerated, but then becomes retarded as heating continues. Lanthanide ions bind to gelatin and collagen. In so doing, they appear to protect these substrates from lysis by clostridiopeptidase through mechanisms additional to supplanting Ca2+ at its binding site on the enzyme. Collagen and gelatin sequester sufficient lanthanide ions to gain partial protection from clostridiopeptidase in the absence of an extraneous source of these inhibitors.

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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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Effect of Interacting Organic Co-solutes with Enzyme Substrate Complex on the Hydrolysis of Raw Soluble Starch with α-amylase: Theory and Experimentation

Advances in Research ◽

10.9734/air/2016/24269 ◽

2016 ◽

Vol 7 (1) ◽

pp. 1-19

Author(s):

Ikechukwu Udema ◽

Abraham Onigbinde

Keyword(s):

Soluble Starch ◽

Substrate Complex ◽

Enzyme Substrate ◽

Hydrolysis Of

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The kinetics of hydrolysis of some extended N-aminoacyl-l-arginine methyl esters by human plasma kallikrein. Evidence for subsites S2 and S3

Biochemical Journal ◽

10.1042/bj2030149 ◽

1982 ◽

Vol 203 (1) ◽

pp. 149-153 ◽

Cited By ~ 7

Author(s):

P R Levison ◽

G Tomalin

Keyword(s):

Human Plasma ◽

Methyl Esters ◽

Plasma Kallikrein ◽

Substrate Complex ◽

Enzyme Substrate ◽

Rate Limiting Step ◽

Kinetics Of Hydrolysis ◽

Rate Limiting ◽

Kinetics Of ◽

Hydrolysis Of

Subsites in the S2-S4 region were identified in human plasma kallikrein. Kinetic constants (kcat., Km) were determined for a series of seven extended N-aminoacyl-L-arginine methyl esters based on the C-terminal sequence of bradykinin (-Pro-Phe-Arg) or (Gly)n-Arg. The rate-limiting step for the enzyme-catalysed reaction was found to be deacylation of the enzyme. It was possible to infer that hydrogen-bonded interactions occur between substrate and the S2-S4 region of kallikrein. Insertion of L-phenylalanine at residue P2 demonstrates that there is also a hydrophobic interaction with subsite S2, which stabilizes the enzyme-substrate complex. The strong interaction demonstrated between L-proline at residue P3 and subsite S3 is of greatest importance in the selectivity of human plasma kallikrein. The purification of kallikrein from Cohn fraction IV of human plasma is described making use of endogenous Factor XIIf to activate the prekallikrein. Kallikreins I (Mr 91 000) and II (Mr 85 000) were purified 170- and 110-fold respectively. Kallikrein I was used for the kinetic work.

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Preferential in vivo accumulation of sn-2,3-diacylglycerols in postheparin plasma of rats

Canadian Journal of Biochemistry ◽

10.1139/o77-159 ◽

1977 ◽

Vol 55 (10) ◽

pp. 1075-1081 ◽

Cited By ~ 9

Author(s):

N. Morley ◽

A. Kuksis ◽

A. G. D. Hoffman ◽

G. Kakis

Keyword(s):

Portal Vein ◽

Lipoprotein Lipase ◽

Hepatic Lipase ◽

Water Interface ◽

Substrate Complex ◽

Enzyme Substrate ◽

Oil Water ◽

Hydrolysis Of

The stereochemical course of in vivo hydrolysis of triacylglycerols by lipoprotein lipase was investigated by determining the structure of diacylglycerol intermediates in postheparin plasma of rats which had been fed [3H]glycerol-labeled Intralipid 2 h before an injection of heparin or had been given an injection of a mixture of [3H]glycerol-Intralipid and heparin. During the clearance of both the natural chylomicrons and the artificial emulsion, sn-2,3-diacylglycerols (60–80%) were found to be the dominant enantiomers. Similar results were obtained when the contribution of the hepatic lipase was altered, either by tying off the mesentery artery and portal vein before injection of heparin, or by injection of heparin directly into the portal vein. These findings are consistent with a preferential release of the acyl group from the sn-1 position of the triacylglycerol molecule as demonstrated previously in vitro. A preferential orientation of the substrate in the enzyme–substrate complex or at the oil–water interface is discussed as a possible basis for these effects.

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015050 ◽

2020 ◽

pp. jbc.RA120.015050

Author(s):

Shrenik C Mehta ◽

Ian M Furey ◽

Orville A Pemberton ◽

David M Boragine ◽

Yu Chen ◽

...

Keyword(s):

Active Site ◽

Substrate Complex ◽

Enzyme Substrate ◽

The Common ◽

Covalent Intermediate ◽

Hydrolysis Of ◽

Carbapenem Antibiotic ◽

Enzyme 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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Site-directed mutagenesis of β-lactamase I. Single and double mutants of Glu-166 and Lys-73

Biochemical Journal ◽

10.1042/bj2720613 ◽

1990 ◽

Vol 272 (3) ◽

pp. 613-619 ◽

Cited By ~ 77

Author(s):

R M Gibson ◽

H Christensen ◽

S G Waley

Keyword(s):

Rate Constants ◽

Double Mutant ◽

Enzyme Mechanism ◽

Site Directed Mutagenesis ◽

Beta Lactamase ◽

Wild Type ◽

Substrate Complex ◽

Enzyme Substrate ◽

Burst Kinetics ◽

Hydrolysis Of

Two single mutants and the corresponding double mutant of beta-lactamase I from Bacillus cereus 569/H were constructed and their kinetics investigated. The mutants have Lys-73 replaced by arginine (K73R), or Glu-166 replaced by aspartic acid (E166D), or both (K73R + E166D). All four rate constants in the acyl-enzyme mechanism were determined for the E166D mutant by the methods described by Christensen, Martin & Waley [(1990) Biochem. J. 266, 853-861]. Both the rate constants for acylation and deacylation for the hydrolysis of benzylpenicillin were decreased about 2000-fold in this mutant. In the K73R mutant, and in the double mutant, the rate constants for acylation were decreased about 100-fold and 10,000-fold respectively. All three mutants also had lowered values for the rate constants for the formation and dissociation of the non-covalent enzyme-substrate complex. The specificities of the mutants did not differ greatly from those of wild-type beta-lactamase, but the hydrolysis of cephalosporin C by the K73R mutant gave ‘burst’ kinetics.

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Synthesis and Kinase Phosphorylation of 4-Deoxy-D-threohexulose

Canadian Journal of Biochemistry ◽

10.1139/o73-125 ◽

1973 ◽

Vol 51 (7) ◽

pp. 969-972 ◽

Cited By ~ 3

Author(s):

Clifford Raymond Haylock ◽

Keith Norman Slessor

Keyword(s):

Lithium Aluminum Hydride ◽

Aluminum Hydride ◽

Kinetic Studies ◽

Lithium Aluminum ◽

Substrate Complex ◽

Acetobacter Suboxydans ◽

Enzyme Substrate ◽

Kinase Phosphorylation ◽

Hydrolysis Of ◽

Yeast Hexokinase

Synthesis of the only unknown deoxyfructose, 4-deoxy-D-threohexulose, is reported. Its preparation involved reductive lithium aluminum hydride ring opening of 3,4-anhydro-1,2:5,6-di-O-isopropylidene- D-talitol, followed by hydrolysis of the resulting epimeric deoxy diisopropylidene hexitols and selective Acetobacter suboxydans oxidation of 3-deoxy-D-arabinohexitol. Kinetic studies using 4-deoxy-D-threohexulose as substrate for yeast hexokinase support the premise that the C-4 hydroxyl is a binding group in formation of the enzyme–substrate complex. Enzymatic synthesis of 4-deoxy-D-threohexulose 6-phosphate and 4-deoxy-D-threohexulose 1,6-diphosphate has been achieved in low yield from 4-deoxy-D-threohexulose.

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