The reactions of sultones with chymotrypsin. The pH dependence of sulfonylation and desulfonylation

Heidema, John H.; Kaiser, E. T.

doi:10.1021/ja01009a031

Cited by 27 publications

(9 citation statements)

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“…Values for human trypsin were obtained from a calibration curve with human duodenal juice compared with data obtained from bovine trypsin. The concentration of active chymotrypsin was determined with 2-hydroxy-5-nitrotoluenesulfoni c acid sulfone (10).…”

Section: Methodsmentioning

confidence: 99%

Isolation and Characterization of a New Trypsin Inhibitor from Crotalaria paulina Seeds

Pando

Ciero²,

Novello

et al. 1999

IUBMB Life

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SummaryA new trypsin inhibitor (CPTI) has been isolated from Crotalaria paulina seeds. Puri cation of the inhibitor was carried out by gel ltration, ion-exchange chromatography, and subsequent reversed-phase HPLC. The presence of a single polypeptide chain, with a molecular mass of 20 kDa and isoelectric point 4.0, was detected. The trypsin inhibitor had a K i value of 4.5 £ 10 ¡ 8 M and was capable of acting on human, bovine, and porcine trypsin and weakly on bovine chymotrypsin. Amino acid analysis showed that CPTI has a high content of aspartate, glutamate, leucine, serine, and glycine, having 177 amino acid residues in its composition. These data suggest that the protein belongs to the Kunitz-type trypsin inhibitors. IUBMB Life, 48: 519-523, 1999

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Section: Methodsmentioning

confidence: 99%

Isolation and Characterization of a New Trypsin Inhibitor from Crotalaria paulina Seeds

Pando

Ciero²,

Novello

et al. 1999

IUBMB Life

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“…3H-1,2-Benzoxathiole 1,1-dioxide was synthesized as described by Heidema and Kaiser (1968). 1 -Chlorotridecan-2-one and 1 -chlorononadec-10-en-2-one were a gift from Professor B.…”

Section: Methodsmentioning

confidence: 99%

Interface-mediated inactivation of pancreatic lipase by a water-reactive compound: 2-sulfobenzoic cyclic anhydride

Moulin¹,

Fourneron²,

Piéroni³

et al. 1989

Biochemistry

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2-Sulfobenzoic cyclic anhydride (SBA) rapidly and selectively inactivates porcine pancreatic lipase (PPL) only when added during the hydrolysis of an emulsified ester such as tributyrin or dodecyl acetate. The present data suggest that the inactivation of PPL occurs preferentially at the oil/water interface and not in the aqueous phase, since colipase and bile salt were found to adversely affect the inhibition process. Moreover, it is shown that at a molar ratio of SBA to pure PPL of 1, 40% of the lipase activity was already irreversibly lost. Complete inactivation was observed at SBA to pure PPL molar ratios of 120. A 60% inactivation occurred when 0.5 mol of 3H-labeled SBA was attached per mole of PPL. The SBA-inactivated PPL competes for binding to the dodecyl acetate/water interface as efficiently as the native enzyme. Larger SBA concentrations are required when crude lipase preparations are used as well as with pure PPL in the presence of bile salts and colipase. Lipases were found to have variable sensitivities to SBA inactivation, depending on their origin. In the presence of bile salts and tributyrin at pH 6.0, human gastric lipase activity was not affected by the presence of a 10(6) molar excess of SBA.

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“…Since the extent of reversibility of step 2 is of conceptual importance in this system, it is appropriate to define the equilibrium in terms of pH-independent rate and equilibrium constants. Furthermore, the unusual pH dependence found for -chymotrypsin-mediated sultone I hydrolysis (Heidema & Kaiser, 1968, 1970), a close structural analogue of sultone II, established a need for a thorough investigation of the kinetic .003…”

Section: Discussionmentioning

confidence: 99%

Energetics of .alpha.-chymotrypsin-mediated hydrolysis of a strained cyclic ester

Bolen¹,

Kimura²,

Nitta³

1987

Biochemistry

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Hydrolysis of omicron-hydroxy-alpha-toluenesulfonic acid sultone (sultone II) is mediated by alpha-chymotrypsin. Sultone II is a highly strained cyclic ester substrate that forms a covalent intermediate with the enzyme and is therefore expected to release ring-strain energy upon formation of the sulfonyl enzyme species. It is found that the equilibrium constant for forming the covalent intermediate from the Michaelis complex is quite modest (K2 = 16.4), suggesting that perhaps the strain energy is not released in the ring-cleavage event. The implied retention of chemical (strain) energy by the sulfonyl enzyme species raises the question of the means by which the enzyme avoids expression of strain energy and the implications of this effect in the catalytic sequence. High-pressure liquid chromatography (HPLC) rate data demonstrate facile reversion of sulfonyl enzyme to the Michaelis complex, and that reversion is preferred over hydrolysis of the covalent intermediate. pH-independent rate and equilibrium constants are derived for the alpha-chymotrypsin-mediated hydrolysis of sultone II, and pKa values for groups on the enzyme are reported that are consistent with literature values obtained from analysis of nonspecific substrate hydrolysis by the enzyme.

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The reactions of sultones with chymotrypsin. The pH dependence of sulfonylation and desulfonylation

Cited by 27 publications

References 0 publications

Isolation and Characterization of a New Trypsin Inhibitor from Crotalaria paulina Seeds

Isolation and Characterization of a New Trypsin Inhibitor from Crotalaria paulina Seeds

Interface-mediated inactivation of pancreatic lipase by a water-reactive compound: 2-sulfobenzoic cyclic anhydride

Energetics of .alpha.-chymotrypsin-mediated hydrolysis of a strained cyclic ester

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