Evidence that the catalytic activity of papain depends on at least four ionizations

Jarvis, Derek; Brocklehurst, Keith

doi:10.1042/bst0100215

Cited by 4 publications

(4 citation statements)

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“…The values of k,[/ K, ' for the papain-catalyzed hydrolyses are in reasonable agreement with those previously reported Lowe & Williams (1965);Z-Gly-ONp, Kirsch & Igelstrom (1966), Williams & Whitaker (1967), Mole & Horton (1973), and Bendall et al (1977Z-Lys-ONp, Bender & Brubacher (1966) and Jarvis & Brocklehurst (1982)]. The relative values of k,,(/K,' for pHNP, Z-Gly-ONp, and Z-Lys-ONp reflect the essential specificity requirements of the enzyme.…”

Section: Discussionsupporting

confidence: 91%

Absence in papaya peptidase A catalyzed hydrolyses of a pKa .apprx. 4 present in papain-catalyzed hydrolyses

Schack¹,

Kaarsholm²

1984

Biochemistry

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Section: Discussionsupporting

confidence: 91%

Absence in papaya peptidase A catalyzed hydrolyses of a pKa .apprx. 4 present in papain-catalyzed hydrolyses

Schack¹,

Kaarsholm²

1984

Biochemistry

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“…Evidence for co-operative ionizations in the free papain molecule has been presented pre-viously (see Brocklehurst, 1982, and references cited therein). The phenomenon discussed in the present paper, in which overlapping ionizations can provide for apparent PKa variation without appreciable profile distortion together with the variations in experimental PKa values reported by Polgar & Halasz (1978), seems to support the suggestions (Shipton & Brocklehurst, 1978;Allen et al, 1978;Jarvis & Brocklehurst, 1982) that the catalytic activity of papain in acidic media might be modulated by ionizations additional to those of the thiol group and the imidazolium ion.…”

supporting

confidence: 90%

“…This is the limit of variability predicted by the simulation study of the model of Scheme 2, and this maximum variation would predict some reactions in which El (of Schemes 2 and 3) is very small relative to k2 and some reactions in which the values of these rate constants (equivalent to pH-independent values of kcat/Km for the acylations) are closely similar. Evidence has accumulated that reactions of the papain thiol group might be modulated by several ionizing groups (see Jarvis & Brocklehurst, 1982, and references cited therein). The remarkable variation in pKapp.…”

Section: Consequences Of Conformational Selectivity For Ph-dependent mentioning

confidence: 99%

Effects of conformational selectivity and of overlapping kinetically influential ionizations on the characteristics of pH-dependent enzyme kinetics. Implications of free-enzyme pKa variability in reactions of papain for its catalytic mechanism

Brocklehurst¹,

Willenbrock²,

Salih³

1983

Biochemical Journal

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The effects of selection by a small molecule, when binding to a protein, of a particular conformation from an equilibrium stereopopulation on the characteristics of the pH-dependence of reaction with a reactivity probe or substrate were determined by analysis of an appropriate kinetic model. For reaction in one protonic state containing an equilibrium mixture of two conformational isomers, the pH-second-order rate constant (k) profile is of conventional sigmoidal form. The apparent pKa value is a composite of the pKa values of the two conformational states. The value of pKapp. for a given enzyme under given experimental conditions will always be the same (provided that the site-specificity assumed in the model is maintained) irrespective of whether only one conformation reacts or both react, with the same or with different rate constants. The experimentally determined pH-independent rate constant (kapp.) is an average of the reactivities of the two conformational states weighted in favour of the predominant form. The presence of an additional but unreactive conformational state also affects the value of kapp. The possibility that overlapping acid dissociations that affect the reactivity of the enzyme might provide pH-k profiles often indistinguishable in practice from simple sigmoidal dissociation curves and subject to variability in apparent pKa values was evaluated by a simulation study. If two reactive protonic states of the enzyme respond differently to changes in the structure of the substrate or site-specific reactivity probe, differences in apparent pKa values of up to approx. 1 unit can be exhibited without deviation from sigmoidal behaviour being reliably observed. Differences in apparent pKa values observed in some site-specific reactions of papain and their possible consequences for its catalytic mechanism are discussed.

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“…In all these studies, as well as the present study, only one acidic ionization and one basic ionization are required to fit the data to the bell-shaped profile. However, there are a few reports in the literature, based on more data points at lower pH values, that provide evidence that for papain-catalyzed ester hydrolysis the acidic limb of the k2/Ks against pH plots is defined by two (Lewis et al, 1978) or three ionizations (Jarvis & Brocklehurst, 1982). Additional evidence for the involvement of more than one ionization comes from studies on papain using two-protonicstate reactivity probes (Brocklehurst & Little, 1972;Shipton & Brocklehurst, 1978).…”

Section: Resultsmentioning

confidence: 99%

Comparison of the kinetics and mechanism of the papain-catalyzed hydrolysis of esters and thiono esters

Storer¹,

Carey²

1985

Biochemistry

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The kinetic constants for the papain-catalyzed hydrolysis of the methyl thiono esters of N-benzoylglycine and N-(beta-phenylpropionyl)glycine are compared with those for the corresponding methyl ester substrates. The k2/Ks values for the thiono esters are 2-3 times higher than those for the esters, and both show bell-shaped pH dependencies with similar pKa's (approximately 4 and 9). The k3 values for the thiono esters are 30-60 times less than those for the esters and do not exhibit a pH dependency. Solvent deuterium isotope effects on k2/Ks and k3 were measured for the ester and thiono ester substrates of both glycine derivatives. Each thiono ester substrate showed an isotope effect similar to that for the corresponding ester substrate. Moreover, use of the proton inventory technique indicated that, as for esters, one proton is transferred in the transition state for deacylation during reactions involving thiono esters and the degree of heavy atom reorganization in the transition state is very similar in both cases. The k3 values for the hydrolysis of a series of para-substituted N-benzoylglycine esters were found to correlate with the k3 values for the corresponding para-substituted thiono esters [Carey, P. R., Lee, H., Ozaki, Y., & Storer, A. C. (1984) J. Am. Chem. Soc. 106, 8258-8262], showing that the rate-determining step for the deacylation of both thiolacyl and dithioacyl enzymes probably involves the disruption of a contact between the substrate's glycinic nitrogen atom and the sulfur of cysteine-25. It is concluded that the hydrolysis of esters and thiono esters proceeds by essentially the same reaction pathway. Due to an oxygen-sulfur exchange process the product released in the case of the N-(beta-phenylpropionyl)glycine thiono ester substrate is the dioxygen acid; however, for the N-benzoylglycine thiono ester substrate, the thiol acid is the initial product. This thiol acid then acts as a substrate for papain and reacylates the enzyme to eventually give the dioxygen acid product. It is shown that thiol acids are excellent substrates for papain.

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Evidence that the catalytic activity of papain depends on at least four ionizations

Cited by 4 publications

References 0 publications

Absence in papaya peptidase A catalyzed hydrolyses of a pKa .apprx. 4 present in papain-catalyzed hydrolyses

Absence in papaya peptidase A catalyzed hydrolyses of a pKa .apprx. 4 present in papain-catalyzed hydrolyses

Effects of conformational selectivity and of overlapping kinetically influential ionizations on the characteristics of pH-dependent enzyme kinetics. Implications of free-enzyme pKa variability in reactions of papain for its catalytic mechanism

Comparison of the kinetics and mechanism of the papain-catalyzed hydrolysis of esters and thiono esters

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