Effects of proflavine on papain's catalytic activity. Proflavine-mediated decrease in KM

215

204

The dependence on thiol pK of the second-order rate constant (kS) for reaction of thiolate anions with MMTS was shown to follow the Brønsted equation log kS = log G + beta pK with log G = 1.44 and 3.54 and beta = 0.635 and 0.309 for aryl and alkyl thiols, respectively. The reactivity toward MMTS of the protonated thiol group was found to be negligible in comparison to that of the thiolate anion. For 2-mercaptoethanol the reactivity toward MMTS of the protonated form of the thiol group was shown to be at least 5 X 10(9) smaller than that of the thiolate anion. The pH dependence of the second-order rate constant for reaction of the thiolate group of Cys-25 at the active site of papain was determined and shown to be consistent with the previously determined low pK for Cys-25 and its electrostatic interaction with His-159. The small dependence of the reactivity of Cys-25 on thiol pK (beta approximately 0.09) suggested that the charge-charge interactions that act through space to perturb the pK of the nucleophile at the active site of papain and perhaps other enzymes may serve to increase the fraction of nucleophile present in the reactive basic form without introducing the decrease in nucleophilic reactivity seen in model systems where pK's are lowered primarily by charge-dipole interactions.

“…determined from the thiol titer. These values were 95% of that estimated from the absorbance at 280 nm by using a molar absorptivity of 5.77 X 104 M"1 cm"1 (Skalski et al, 1973).…”

Section: Methodsmentioning

confidence: 58%

Reactivity of small thiolate anions and cysteine-25 in papain toward methyl methanethiosulfonate

Roberts¹,

Lewis²,

Ballou³

et al. 1986

215

204

“…Concentrations of papain and derivatives of papain were determined from the absorbance at 280 nm with a molar absorptivity of 5.77 X 104 M"1 cm-1 (Skalski et al, 1973). The catalytic activity of papain was routinely assayed by one of two methods.…”

Section: Methodsmentioning

confidence: 99%

Determination of a low pK for histidine-159 in the S-methylthio derivative of papain by proton nuclear magnetic resonance spectroscopy

Johnson¹,

Lewis²,

Shafer³

1981

Proton NMR spectroscopy was used to study the ionization behavior of His-159 in a derivative of papain (papain-S-SCH3). In this catalytically inactive derivative of papain, the active-site thiol group of Cys-25 is S-methyl-thiolated so that it cannot form a thiolate anion. The pH dependence of the chemical shift of the C epsilon 1 H resonance of His-159 indicated a pK of 3.45 +/- 0.07 at 45 degrees C in 2H2O with no added ions other than those required for titration. In acetate buffers at an ionic strength of 0.05, the pK increased to 3.87 +/- 0.12. Conversion of papain-S-SCH3 to active papain at pH* 4.17 (at 45 degrees C and an ionic strength of 0.05) caused the position of the C epsilon 1 H resonance to change from a position indicative of partial protonation of His-159 to a position indicative of full protonation, consistent with the existence of an imidazolium-thiolate ion-pair interaction between His-159 and Cys-25 in the active enzyme.

“…Concentrations of papain and derivatives of papain were determined from the absorbance at 280 nm using a molar absorptivity of 5.77 X 104 cm-1 M-1 (Skalski et al, 1973). Using this value for the molar absorptivity of papain, the thiol titer of the papain used in this work was 0.96 as determined from the increase in absorbance at 420 nm when 1 mM Nbs2 and 0.05 mM papain were mixed at pH 7.2.…”

Section: Methodsmentioning

confidence: 99%

Potentiometric determination of ionizations at the active site of papain

Lewis¹,

Johnson²,

Shafer³

1976

140

120

The ionization behavior of groups at the active site of papain was determined from the pH dependence of the difference of proton content of papain and the methylthio derivative of the thiol group at the active site of papain (papain-S-SCH3). This difference in proton content was determined directly by two independent methods. One method involved potentiometric measurements of the protons released and demethylthiolation of papain-S-SCH3 with dithiothreitol, as a function of pH. The other method involved analogous measurements of the protons released on methylthiolation of papain with methyl methanethiosulfonate. The methylthio pH-difference titrations generated by these measurements indicate that ionization of the thiol group at the active site of papain is linked to the ionization of His-159. The pK of the thiol group changes from 3.3 to 7.6 on deprotonation of His-159 at 29 degrees C/20.05. Similarly, the pK of His-159 shifts from 4.3 to 8.5 when the active site thiol group is deprotonated. The microscopic ionization constants determined in this work for Cys-25 and His-159 indicate that equilibrium constant for transfer of the proton from Cys-25 to His-159 is 8--12, and that in the physiological pH range the active site thiol group exists mainly as a thiol anion.