.alpha.-Chymotrypsin-catalyzed hydrolysis of lactones

Tobias, Peter S.; Heidema, John H.; Lo, Kwok-Wing; Kaiser, E. T.; Kézdy, Ferenc J.

doi:10.1021/ja01029a043

Cited by 31 publications

(6 citation statements)

References 3 publications

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“…The nitrolactones used here were made by nitration of the commercially available precursors with a standard nitration method as used in the synthesis of DMNB [15]. 5-NC and 6-NDC had a melting point of 187-189 and 134-136 mC respectively ; Tobias et al [16] give 189-189.5 and 130-131 mC. The identities of 5-NC and 6-NDC were confirmed by NMR spectrometry.…”

Section: Experimental Materialsmentioning

confidence: 99%

“…The esters and their corresponding lactones cannot necessarily be expected to behave identically. The lactones must be ' cisoid ' in configuration, whereas the non-cyclic esters are free to adopt a ' transoid ' configuration [16] ; similar differences in shape are shown by DMNB and its non-cyclic analogue, PNP dimethylcarbamate [26]. The lactones are intrinsically more reactive than the esters (for example, to spontaneous hydrolysis) ; this may be partly a steric effect and partly an electronic one in that resonance stabilization of the following kind may not be as significant when the grouping is part of a 5-or 6-membered ring :…”

Section: Effect Of Nad + On Hydrolysis Of 5-nc and 6-ndcmentioning

confidence: 99%

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A comparison of nitrophenyl esters and lactones as substrates of cytosolic aldehyde dehydrogenase

Kitson

1996

Biochemical Journal

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1. p-Nitrophenyl (PNP) acetate and propionate show a burst of p-nitrophenoxide release when their hydrolysis is catalysed by sheep liver cytosolic aldehyde dehydrogenase. This is not seen in the presence of NAD+ or NADH, implying a change in ratedetermining step. 2. 6-Nitrodihydrocoumarin (6-NDC) shows no burst of absorbance in the visible region. We propose that the pKa of the transient "reporter group' produced during the hydrolysis of this lactone is high (approx. 10) and that the incipient covalently linked p-nitrophenoxide moiety is protonated immediately on formation. The small burst seen in the hydrolysis of 5-nitro-2-coumaranone (5-NC) suggests that the pKa of its reporter group is about 8.5. 3. NADH markedly enhances the steady-state rate with the lactones. 5-NC shows a large rapid burst of colour development in the presence of NADH; this implies that NADH decreases the pKa of the reporter group to 7-7.5. 4. In the presence of NAD+, 5-NC and 6-NDC give an unusual "negative burst' in the stopped-flow traces. We propose that, under these circumstances, acylation of the enzyme is extremely fast and that the first event seen in the stopped-flow traces is protonation of the reporter group. NAD+ also greatly increases the steady-state rate. 5. With the lactones in the presence of NADH, the kcat value (nearly 6 s-1), a measure of the deacylation rate, is compatible with the single-site model for dehydrogenase and esterase activities.

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Section: Experimental Materialsmentioning

confidence: 99%

Section: Effect Of Nad + On Hydrolysis Of 5-nc and 6-ndcmentioning

confidence: 99%

A comparison of nitrophenyl esters and lactones as substrates of cytosolic aldehyde dehydrogenase

Kitson

1996

Biochemical Journal

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“…One possibility for reverting the inactivation process is the incorporation of an intramolecular nucleophile that can catalyze dephosphonylation. Kaiser's group (Kaiser et al, 1971;Heidema & Kaiser, 1967;Tobias et al, 1969) reported reversible inhibition of serine proteases by acyl, sulfonyl and phosphoryl compounds that contained a /3-hydroxy (phenolic) nucleophile for intramolecular reactivation of enzyme activity. The inactivation rates by these compounds were quite slow, but the reactivation rates were even slower.…”

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confidence: 99%

Enantioselective and Reversible Inhibition of Trypsin and .alpha.-Chymotrypsin by Phosphonate Esters

Zhao¹,

Kovach²,

Bencsura³

et al. 1994

Biochemistry

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Trypsin is inactivated by the levorotatory enantiomers (most likely PS) of 4-nitrophenyl 4-H-, 4-CH3-,4-OCH3-, and 4-Cl-phenacyl methylphosphonates (PMNs) with second-order rate constants between 231 and 884 M-1 s-1. 4-NO2-PMN hydrolyzes before inhibiting the enzyme. The second-order rate constants for the inactivation of alpha-chymotrypsin by the levorotatory enantiomers of the five PMNs are between 37,000 and 770,000 M-1 s-1, and those for the dextrorotatory enantiomers are between 400 and 640 M-1 s-1; the enantioselectivity is 90-1880. Specific rotation [alpha]22D of the faster-reacting enantiomer of 4-CH3-PMN with trypsin and alpha-chymotrypsin is -30 +/- 6 degrees. 31P NMR of the adducts shows a signal at 41.0 ppm, 10 ppm downfield from the parent compound. Results of molecular mechanics and dynamics calculations show that the principal interactions are between the phosphonyl group and constituents of the oxyanion hole and between the aromatic fragment and residues in the binding regions of the enzymes. Trypsin activity returns from its phenacyl methylphosphonyl adducts on the hour time scale and in reversed order to the rates of inactivation within the series. Recovery of alpha-chymotrypsin activity from the adducts formed with the (-) enantiomers is on a slower time scale still, whereas its recovery from the adducts formed with the (+) enantiomers is on the second to minute time scale. The data support a mechanism of reactivation involving rate-determining intramolecular displacement of Ser by the carbonyl hydrate of the phenacyl moiety. The pH-rate profiles for trypsin reactivation from its adducts indicate involvement of an ionizable group with pKa approximately 8.0. The pH dependence and solvent isotope effects are small in most cases. The compounds demonstrate favorable properties for controllable and temporary modulation of enzyme activity.

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“…Known alkylating inhibitors may be further classified as those that are intrinsically electrophilic and those in which the electrophilic functionality is initially masked and exposed only 1 Abbreviations: HEPES, N-(2-hydroxyethyl)piperazine-N'-2ethanesulfonic acid; HLE, human leukocyte elastase; HSE, human sputum elastase; MES, 2-(N-morpholino)ethanesulfonic acid; Me2SO, dimethyl sulfoxide; PPE, porcine pancreatic elastase; SAAPVC, 7-(methoxysuccinylalanylalanylprolylvalinamido)-4-methylcoumarin; SAAPVFC, 7-(methoxysuccinylalanylalanylprolylvalinamido)-4-(trifluoromethyl)coumarin; TAPS, 3-[ [tris(hydroxymethyl)methyl] amino]propanesulfonic acid; TMAC, 7-[(trimethylacetyl)oxy]-4-methylcoumarin (see Materials and Methods). 2 The lactones of Tobias et al (1969) and Izbicka and Bolen (1981) are an interesting exception. Though these compounds acylate chymotrypsin, they can deacylate intramolecularly to regenerate the initial inhibitor.…”

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confidence: 99%

Kinetics and mechanism of human leukocyte elastase inactivation by ynenol lactones

Copp¹,

Krantz²,

Spencer³

1987

Biochemistry

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Human leukocyte elastase (HLE), a serine protease involved in inflammation and tissue degradation, can be irreversibly inactivated in a time- and concentration-dependent manner by ynenol lactones. Ynenol lactones that are alpha-unsubstituted do not inactivate but are alternate substrate inhibitors that are hydrolyzed by the enzyme. Ynenol lactones that are both substituted alpha to to the lactone carbonyl and unsubstituted at the acetylene terminus are rapid inactivators of HLE and inactivate pancreatic elastase and trypsin more slowly. 3-Benzyl-5(E)-(prop-2-ynylidene)tetrahydro-2-furanone inactivates HLE with biphasic kinetics and an apparent second-order rate of up to 22,000 M-1 s-1 (pH 7.8, 25 degrees C). The rate of inactivation is pH-dependent and is slowed by a competitive inhibitor. The partition ratio is 1.6 +/- 0.1. Rapid removal of ynenol lactone during the course of inactivation yields a mixture of acyl and inactivated enzyme species, which then shows a partial recovery of activity that is time- and pH-dependent. Inactivation is not reversible with hydroxylamine. The enzyme is not inactivated if the untethered allenone is added exogenously. All of these results are consistent with a mechanism involving enzyme acylation at serine-195 by the ynenol lactone, isomerization of the acyl enzyme to give a tethered allenone, and capture of a nucleophile (probably histidine-57) to inactivate the enzyme. Substitution at the acetylene terminus of ynenol lactones severely reduces their ability to inactivate HLE, because allenone formation is slowed and/or nucleophile capture is hindered. Chemical competence of each of these steps has been demonstrated [Spencer, R.W., Tam, T.F., Thomas, E.M., Robinson, V.J.,& Krantz, A. (1986) J. Am. Chem. Soc. 108, 5589-5597].

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.alpha.-Chymotrypsin-catalyzed hydrolysis of lactones

Abstract: The a-Chymotrypsin-Catalyzed Hydrolysis of Lactones1 Sir:«-Chymotrypsin, a pancreatic protease and esterase, was recently shown to react rapidly and stoichiometrically with aromatic five-membered sultones.2 The

Cited by 31 publications

References 3 publications

A comparison of nitrophenyl esters and lactones as substrates of cytosolic aldehyde dehydrogenase

A comparison of nitrophenyl esters and lactones as substrates of cytosolic aldehyde dehydrogenase

Enantioselective and Reversible Inhibition of Trypsin and .alpha.-Chymotrypsin by Phosphonate Esters

Kinetics and mechanism of human leukocyte elastase inactivation by ynenol lactones

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