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

2004 ◽  
Vol 384 (2) ◽  
pp. 437-447 ◽  
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.

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

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.

scholarly journals 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

1987 ◽  
Vol 244 (3) ◽  
pp. 553-558 ◽  
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.

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

1980 ◽  
Vol 45 (2) ◽  
pp. 427-434 ◽  
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.

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

1975 ◽  
Vol 53 (7) ◽  
pp. 747-757 ◽  
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.

Human cholinesterases

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.

scholarly journals Role of Sulfated Tyrosines of Thyroglobulin in Thyroid Hormonosynthesis

Endocrinology ◽  
2005 ◽  
Vol 146 (11) ◽  
pp. 4834-4843 ◽  
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.

scholarly journals His224 Alters the R2 Drug Binding Site and Phe218 Influences the Catalytic Efficiency of the Metallo-β-Lactamase VIM-7

2014 ◽  
Vol 58 (8) ◽  
pp. 4826-4836 ◽  
Author(s):  
Hanna-Kirsti S. Leiros ◽  
Susann Skagseth ◽  
Kine Susann Waade Edvardsen ◽  
Marit Sjo Lorentzen ◽  
Gro Elin Kjæreng Bjerga ◽  
...  
Keyword(s):  
Hydrogen Bonding ◽  
Active Site ◽  
Pathogenic Bacteria ◽  
Catalytic Efficiency ◽  
Drug Binding ◽  
Side Chain ◽  
Wild Type ◽  
Drug Binding Site ◽  
Chain Conformations ◽  
Positively Charged

ABSTRACTMetallo-β-lactamases (MBLs) are the causative mechanism for resistance to β-lactams, including carbapenems, in many Gram-negative pathogenic bacteria. One important family of MBLs is the Verona integron-encoded MBLs (VIM). In this study, the importance of residues Asp120, Phe218, and His224 in the most divergent VIM variant, VIM-7, was investigated to better understand the roles of these residues in VIM enzymes through mutations, enzyme kinetics, crystal structures, thermostability, and docking experiments. The tVIM-7-D120A mutant with a tobacco etch virus (TEV) cleavage site was enzymatically inactive, and its structure showed the presence of only the Zn1 ion. The mutant was less thermostable, with a melting temperature (Tm) of 48.5°C, compared to 55.3°C for the wild-type tVIM-7. In the F218Y mutant, a hydrogen bonding cluster was established involving residues Asn70, Asp84, and Arg121. The tVIM-7-F218Y mutant had enhanced activity compared to wild-type tVIM-7, and a slightly higherTm(57.1°C) was observed, most likely due to the hydrogen bonding cluster. Furthermore, the introduction of two additional hydrogen bonds adjacent to the active site in the tVIM-7-H224Y mutant gave a higher thermostability (Tm, 62.9°C) and increased enzymatic activity compared to those of the wild-type tVIM-7. Docking of ceftazidime in to the active site of tVIM-7, tVIM-7-H224Y, and VIM-7-F218Y revealed that the side-chain conformations of residue 224 and Arg228 in the L3 loop and Tyr67 in the L1 loop all influence possible substrate binding conformations. In conclusion, the residue composition of the L3 loop, as shown with the single H224Y mutation, is important for activity particularly toward the positively charged cephalosporins like cefepime and ceftazidime.

Destabilization is as important as binding

1993 ◽  
Vol 345 (1674) ◽  
pp. 3-10 ◽  
Keyword(s):  
Binding Energy ◽  
Gibbs Energy ◽  
Active Site ◽  
Electrostatic Interactions ◽  
Large Fraction ◽  
Geometric Distortion ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Non Covalent Interactions ◽  
Covalent Interactions

Enzymes make use of non-covalent interactions with their substrates to bring about a large fraction of their catalytic activity. These interactions must destabilize, or increase the Gibbs energy, of the substrate in the active site in order that the transition state can be reached easily. This destabilization may be brought about by utilization of the intrinsic binding energy between the active site and the bound substrate by desolvation of charged groups, geometric distortion, electrostatic interactions and, especially, loss of entropy in the enzyme-substrate complex. These mechanisms are described by interaction energies and require utilization of the intrinsic binding energy that is realized from non-covalent interactions between the enzyme and substrate. Receptors and coupled vectorial processes, such as muscle contraction and active transport, utilize binding energy similarly to avoid large peaks and valleys along the Gibbs energy profile of the reaction under physiological conditions.

Essential Arginine Residues in Lactate Dehydrogenase from Germinating Soybean

1994 ◽  
Vol 59 (2) ◽  
pp. 467-472 ◽  
Author(s):  
Jana Barthová ◽  
Irena Hulová ◽  
Miroslava Birčáková
Keyword(s):  
Enzyme Activity ◽  
Glycine Max ◽  
Lactate Dehydrogenase ◽  
Chemical Modification ◽  
Active Site ◽  
Enzyme Modification ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Arginine Residues ◽  
Blue Sepharose

The lactate dehydrogenase was isolated from soybean (Glycine max. L.) by a procedure that employed biospecific chromatography on a column of Blue-Sepharose CL-6B. The participation of the guanidine group of arginine residues in the mechanism of enzyme action was determined through kinetic and chemical modification studies. The dependence of enzyme activity on pH was followed in the alkaline region (pH 8.6 - 12.8). The pK values found were 12.4 for the enzyme substrate complex and 11.1 for the free enzyme. The enzyme was inactivated by phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and p-hydroxyphenylglyoxal reagents used in modification experiments. Kinetic analysis of the modification indicated that one arginine residue is modified when inactivation occurs. No effect was observed on the rate of inactivation upon addition of coenzyme. The extent of enzyme modification by p-hydroxyphenylglyoxal was determined. It appears there are at least two arginine residues in the active site of the enzyme.

KINETICS AND MECHANISMS OF REACTIONS CATALYZED BY PANCREATIC RIBONUCLEASE

1966 ◽  
Vol 44 (22) ◽  
pp. 2597-2610 ◽  
Author(s):  
Eileen N. Ramsden ◽  
Keith J. Laidler
Keyword(s):  
Complex Formation ◽  
Active Site ◽  
Histidine Residue ◽  
Aspartic Acid Residue ◽  
Pyrimidine Base ◽  
Substrate Complex ◽  
Imidazole Group ◽  
Enzyme Substrate ◽  
Subsequent Hydrolysis ◽  
Mechanisms Of Reactions

A kinetic study has been made of the ribonuclease-catalyzed hydrolyses of three cyclic nucleotides, cytidine-2′,3′-phosphate, uridine-2′,3′-phosphate, and N6,O5′-diacetyl cytidine-2′,3′-phosphate. Rates were measured at pH values ranging from 6 to 8.5. The variation of the kinetic parameters with pH showed that the free enzyme possesses two active groups, having pK values of 5.4 and 7.25. When the enzyme–substrate complex is formed, the pK values of the groups are increased to 6.6 and 8.4. The pK values identify these groups as imidazole groups and show that two histidine residues are present at the active site. Since both increase in pK on complex formation, it is concluded that the acid imidazole group binds the substrate, but that the basic imidazole group cannot be concerned in substrate binding and must function only in the hydrolytic step. The results indicate that the pyrimidine base is concerned in the hydrolytic step and not solely in binding, as had been postulated. It is concluded from all of the evidence that four specific sites are present at the active center of the enzyme; three are involved in binding and one in catalysis. It is proposed that the active site of ribonuclease is composed of: the histidine residue in position 12, which catalyzes the hydrolytic step; the histidine residue in position 119, which binds the 2′-ribose oxygen atom in the substrate; the lysine residue in position 41, which binds the phosphate group or anion; and the aspartic acid residue in position 121, which binds the nitrogen atom at N1 in the pyrimidine base. A mechanism for enzyme–substrate complex formation and subsequent hydrolysis is proposed.

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