Physicochemical Property Modification Strategies Based on Enzyme Substrate Specificities II: α‐Chymotrypsin Hydrolysis of Aspirin Derivatives

1981 ◽  
Vol 70 (12) ◽  
pp. 1304-1306 ◽  
Author(s):  
Pradip K. Banerjee ◽  
Gordon L. Amidon
Keyword(s):  
Physicochemical Property ◽  
Substrate Specificities ◽  
Enzyme Substrate ◽  
Property Modification ◽  
Hydrolysis Of

scholarly journals Evidence for the regulation of guinea-pig heart microsomal phosphatidylcholine-hydrolysing phospholipase A1 by guanosine 5′-[γ-thio]triphosphate

1992 ◽  
Vol 288 (3) ◽  
pp. 965-968 ◽  
Author(s):  
K Badiani ◽  
X Lu ◽  
G Arthur
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Guinea Pig ◽  
Molecular Species ◽  
Inhibitory Effect ◽  
Phospholipase A1 ◽  
Guinea Pig Heart ◽  
Substrate Specificities ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of

We have recently characterized lysophospholipase A2 activities in guinea-pig heart microsomes and postulated that these enzymes act sequentially with phospholipases A1 to release fatty acids selectively from phosphatidylcholine (PC) and phosphatidylethanolamine, thus providing an alternative route to the phospholipase A2 mode of release. In a further investigation of the postulated pathway, we have characterized the PC-hydrolysing phospholipase A1 in guinea-pig heart microsomes. Our results show that the enzyme may have a preference for substrates with C16:0 over C18:0 at the sn-1 position. In addition, although the enzyme cleaves the sn-1 fatty acid, the rate of hydrolysis of PC substrates with C16:0 at the sn-1 position was influenced by the nature of the fatty acid at the sn-2 position. The order of decreasing preference was C18:2 > C20:4 = C18:1 > C16:0. The hydrolyses of the molecular species were differentially affected by heating at 60 degrees C. An investigation into the effect of nucleotides on the activity of the enzyme showed that guanosine 5′-[gamma-thio]triphosphate (GTP[S]) inhibited the hydrolysis of PC by phospholipase A1 activity, whereas GTP, guanosine 5′-[beta-thio]diphosphate (GDP[S]), GDP, ATP and adenosine 5′-[gamma-thio]triphosphate (ATP[S]) did not affect the activity. The inhibitory effect of GTP[S] on phospholipase A1 activity was blocked by preincubation with GDP[S]. A differential effect of GTP[S] on the hydrolysis of different molecular species was also observed. Taken together, the results of this study suggest the presence of more than one phospholipase A1 in the microsomes with different substrate specificities, which act sequentially with lysophospholipase A2 to release linoleic or arachidonic acid selectively from PC under resting conditions. Upon stimulation and activation of the G-protein, the release of fatty acids would be inhibited.

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.

scholarly journals A rapid direct assay for the determination of the separate activities of the three arylsulphatases of Aspergillus oryzae

1977 ◽  
Vol 166 (3) ◽  
pp. 411-413 ◽  
Author(s):  
G R J Burns ◽  
C H Wynn
Keyword(s):  
Phenolic Compounds ◽  
Aspergillus Oryzae ◽  
Substrate Specificities ◽  
Direct Assay ◽  
Assay Procedure ◽  
Rate Of Hydrolysis ◽  
Hydrolysis Of ◽  
Potential Use

1. The three arylsulphatases of Aspergillus oryzae exhibit pronounced kinetic differences and substrate specificities. Arylsulphatase I hydrolyses all substrates tested, whereas arylsulphatase III will not hydrolyse tyrosine O-sulphate or phenolphthalein disulphate. Arylsulphatase II does not hydrolyse p-nitrophenyl sulphate or phenolphthalein disulphate at appreciable rates in the absence of added phenolic compounds. Phenols such as tyramine increase the rate of hydrolysis of these substances by this enzyme 1000-fold. At pH 6.9 arylsulphatase I exhibits an apparent Km of 0.1 mM for p-nitrophenyl sulphate, whereas the Km of arylsulphatase III for this substrate is 1 mM. 2. These differences were utilized to develop an assay procedure which can be used to determine the separate activities of the three enzymes present in mixtures. This assay has potential use as a means of examining the relative activities of the three enzymes in investigations of the differences in the mechanisms regulating their synthesis.

scholarly journals The stereochemical course of reactions catalysed by the cellobiohydrolases produced by Talaromyces emersonii

1992 ◽  
Vol 283 (1) ◽  
pp. 31-34 ◽  
Author(s):  
M M Brooks ◽  
M G Tuohy ◽  
A V Savage ◽  
M Claeyssens ◽  
M P Coughlan
Keyword(s):  
Degradation Product ◽  
Proteolytic Degradation ◽  
Substrate Specificities ◽  
Talaromyces Emersonii ◽  
Anomeric Configuration ◽  
Culture Filtrates ◽  
Apparent Homogeneity ◽  
Displacement Reactions ◽  
Hydrolysis Of

Three forms of exocellobiohydrolase (EC 3.2.1.91), CBH IA, CBH IB and CBH II, were isolated to apparent homogeneity from culture filtrates of the aerobic fungus Talaromyces emersonii. CBH IA and CBH II appear to be native forms of these enzymes, while CBH IB may represent a proteolytic degradation product of the CBH IA enzyme. The hydrolysis of beta-cellobiosyl fluoride by each form was monitored by 1H-n.m.r. spectroscopy. The reactions catalysed by CBH IA and CBH IB proceed with retention of the anomeric configuration, whereas that catalysed by CBH II is one of inversion. Thus one may deduce that CBH IA (or CBH IB) and CBH II operate double and single displacement reactions respectively during catalysis of substrate. On the basis of these findings and the observed substrate specificities of the various forms, one may conclude that CBH IA (and CBH IB) is a family C enzyme, while CBH II belongs to family B [Henrissat, Claeyssens, Tomme, Lemesle & Mornon (1989) Gene 81, 83-95].

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 Common-type acylphosphatase: steady-state kinetics and leaving-group dependence

1997 ◽  
Vol 327 (1) ◽  
pp. 177-184 ◽  
Author(s):  
Paolo PAOLI ◽  
Paolo CIRRI ◽  
Lucia CAMICI ◽  
Giampaolo MANAO ◽  
Gianni CAPPUGI ◽  
...  
Keyword(s):  
Inorganic Phosphate ◽  
Strong Dependence ◽  
Common Type ◽  
Enzyme Active Site ◽  
Enzyme Substrate ◽  
Linear Relationships ◽  
Steady State Kinetics ◽  
Spontaneous Hydrolysis ◽  
Leaving Group ◽  
Hydrolysis Of

A number of acyl phosphates differing in the structure of the acyl moiety (as well as in the leaving-group pKa of the acids produced in hydrolysis) have been synthesized. The Km and Vmax values for the bovine common-type acylphosphatase isoenzyme have been measured at 25 °C and pH 5.3. The values of kcat differ widely in relation to the different structures of the tested acyl phosphates: linear relationships between log kcat and the leaving group pKa, as well as between log kcat/Km and the leaving-group pKa, were observed. On the other hand, the Km values of the different substrates are very close to each other, suggesting that the phosphate moiety of the substrate is the main chemical group interacting with the enzyme active site in the formation of the enzyme–substrate Michaelis complex. The enzyme does not catalyse transphosphorylation between substrate and concentrated nucleophilic acceptors (glycerol and methanol); nor does it catalyse H218O–inorganic phosphate oxygen exchange. It seems that no phosphoenzyme intermediate is formed in the catalytic pathway. Furthermore, during the enzymic hydrolysis of benzoyl phosphate in the presence of 18O-labelled water, only inorganic phosphate (and not benzoate) incorporates 18O, suggesting that no acyl enzyme is formed transiently. All these findings, as well as the strong dependence of kcat upon the leaving group pKa, suggest that neither a nucleophilic enzyme group nor general acid catalysis are involved in the catalytic pathway. The enzyme is competitively inhibited by Pi, but it is not inhibited by the carboxylate ions produced during substrate hydrolysis, suggesting that the last step of the catalytic process is the release of Pi. The activation energy values for the catalysed and spontaneous hydrolysis of benzoyl phosphate have been determined.

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