Clarification of the pH-dependent kinetic behaviour of papain by using reactivity probes and analysis of alkylation and catalysed acylation reactions in terms of multihydronic state models: implications for electrostatics calculations and interpretation of the consequences of site-specific mutations such as Asp-158-Asn and Asp-158-Glu

1. The complex behaviour of papain (EC 3.4.22.2) in acidic media has been investigated by (a) stopped-flow reactivity probe kinetics using 4,4′-dipyrimidyl disulphide (I) and 2,2′-dipyridyl disulphide (II) as thiol-specific time-dependent inhibitors with markedly different susceptibilities to activation by hydronation (protonation) and (b) using the multitasking application program SKETCHER for the rapid evaluation of pH-dependent kinetic data by means of interactive manipulation of calculated curves. 2. The substantially lower basicity of (I) (pKa 0.91) than that of (II) (pKa 2.45) combined with retention of high reactivity permitted the pKa for the formation of the (Cys-25)-S-/(His-159)-Im+H ion-pair state of papain to be determined kinetically as 3.4, a value close to that (3.3) deduced by potentiometric difference titration [Lewis, Johnson and Shafer (1976) Biochemistry 15, 5009-5017] and lower than the value (approx. 4) often reported from pH-dependent kinetic studies. The higher values are now known to arise from inadequate data analysis that does not take account of other overlapping kinetically influential ionizations. 3. Re-evaluation of the extensive sets of pH-kcat/Km data for the hydrolysis of nine substrates by papain reported by Polgár and Halász (1978) (Eur. J. Biochem. 88, 513-521) by making use of SKETCHER, the known pKa value (3.4) from the reaction with compound (I) and two additional kinetically influential pKa values deduced from the reaction with compound (II) now permits the identification of the pH-dependent events in reactions of papain with inhibitors and substrates. 4. A major conclusion is that, whereas in reactions of simple alkylating agents and compound (I) full nucleophilic character of (Cys-25)-S-/(His-159)-Im+H is provided by hydronic dissociation with pKa 3.3-3.4, in catalysis relatively little catalytic competence is produced consequent upon ion-pair formation. Substantial catalytic competence requires further hydronic dissociation with pKa approx. 4, and for cationic substrates further enhancement is produced by hydronic dissociation with pKa approx. 5. 5. The present work, together with the kinetic analysis of reactions of papain in alkaline media reported by Mellor, Thomas, Topham and Brocklehurst [Biochem. J. (1993) 290, 289-296], defines the kinetically influential ionizations of papain as 3.4, 4.0, 5.0, 8.3 and 10.0 of which 3.4 and 8.3 relate to the formation and subsequent dehydronation of the ion-pair state.(ABSTRACT TRUNCATED AT 400 WORDS)

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Ionization characteristics of the Cys-25/His-159 interactive system and of the modulatory group of papain: resolution of ambiguity by electronic perturbation of the quasi-2-mercaptopyridine leaving group in a new pyrimidyl disulphide reactivity probe

Biochemical Journal ◽

10.1042/bj2900289 ◽

1993 ◽

Vol 290 (1) ◽

pp. 289-296 ◽

Cited By ~ 22

Author(s):

G W Mellor ◽

E W Thomas ◽

C M Topham ◽

K Brocklehurst

Keyword(s):

Ph Dependence ◽

Order Rate Constant ◽

Ion Pair ◽

Catalytic Site ◽

Alkaline Media ◽

Interactive System ◽

Data Set ◽

Pka Value ◽

Compound Ii ◽

Pair State

1. A new thiol-specific reactivity probe 4,4′-dipyrimidyl disulphide [compound (VII), m.p. 110 degrees C, pKa of its monohydronated form 0.91] was synthesized and used to resolve the ambiguity of interpretation of the behaviour of papain (EC 3.4.22.2) in alkaline media known to depend to varying extents on two ionizations with pKa values approx. 8.0-8.5 and > or = 9.5 respectively. 2. A new extensive pH-second-order rate constant (k) data set for the reaction of papain with 2-(acetamido)-ethyl 2′-pyridyl disulphide (IV) demonstrated the existence of a striking rate maximum at pH approx. 4, the independence of k around pH 8 and the increase in k with increase in pH across a pKa value of 10.0, behaviour similar to that of other 2-pyridyl disulphides (R-S-S-2-Py) that lack key substrate-like binding sites in R. 3. Although the simplest interpretation of the pKa value of 10.0 assigns it to the formation of (Cys-25)-S-/(His-159)-Im from the ion-pair state of the papain catalytic site, another interpretation may be conceived in which this pKa value is assigned to another group remote from the catalytic site, the state of ionization of which modulates catalytic-site behaviour. This alternative assignment is shown to require compensating effects in the pH region around 8 such that the formation of (Cys-25)-S-/(His-159)-Im across pKa 8.0-8.5 is without net kinetic effect in the reactions of simple 2-pyridyl disulphides such as compound (IV) and 2,2′-dipyridyl disulphide (II). 4. The lower basicity of compound (VII) relative to that of compound (II) (pKa 2.45) was predicted to diminish or abolish the compensation postulated as a possibility in reactions of 2-pyridyl disulphides because of the decreased effectiveness of reaction via a (His-159)-Im+H-assisted transition state. The characteristics of the pH-dependence of the reaction of papain with compound (VII) which are quite different from those for its reaction with compound (II) support both this prediction and the alternative assignment with a value of 8.3 for the pKa of the formation of (Cys-25)-S-/(His-159)-Im. 5. Evidence that the behaviour of papain towards both substrates and some substrate-derived time-dependent inhibitors is determined not only by the loss of the (Cys-25)-S-/(His-159)-Im+H ion-pair state by dehydronation with pKa 8.3 but also by another ionization of pKa approx. 10.0 is briefly discussed.

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Kinetic studies on the ion-pair state in the E region of iodine in collision with a N2 gas molecule

Chemical Physics Letters ◽

10.1016/0009-2614(92)85943-5 ◽

1992 ◽

Vol 196 (1-2) ◽

pp. 139-144 ◽

Cited By ~ 5

Author(s):

Zhi-yuan Min ◽

Chong-de Li ◽

Jia-ming Wang ◽

Shun-sheng Gong

Keyword(s):

Kinetic Studies ◽

Ion Pair ◽

Gas Molecule ◽

E Region ◽

Pair State

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pH-Dependent Thermal Stability of Vibrio cholerae L-asparaginase

Protein and Peptide Letters ◽

10.2174/0929866526666190617092944 ◽

2019 ◽

Vol 26 (10) ◽

pp. 743-750 ◽

Cited By ~ 1

Author(s):

Remya Radha ◽

Sathyanarayana N. Gummadi

Keyword(s):

Vibrio Cholerae ◽

Conformational Changes ◽

Kinetic Studies ◽

Structural Features ◽

Structure Stability ◽

Kcal Mole ◽

Scanning Calorimetry ◽

Wide Range ◽

Ph Dependent ◽

Ph Range

Background:pH is one of the decisive macromolecular properties of proteins that significantly affects enzyme structure, stability and reaction rate. Change in pH may protonate or deprotonate the side group of aminoacid residues in the protein, thereby resulting in changes in chemical and structural features. Hence studies on the kinetics of enzyme deactivation by pH are important for assessing the bio-functionality of industrial enzymes. L-asparaginase is one such important enzyme that has potent applications in cancer therapy and food industry.Objective:The objective of the study is to understand and analyze the influence of pH on deactivation and stability of Vibrio cholerae L-asparaginase.Methods:Kinetic studies were conducted to analyze the effect of pH on stability and deactivation of Vibrio cholerae L-asparaginase. Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) studies have been carried out to understand the pH-dependent conformational changes in the secondary structure of V. cholerae L-asparaginase.Results:The enzyme was found to be least stable at extreme acidic conditions (pH< 4.5) and exhibited a gradual increase in melting temperature from 40 to 81 °C within pH range of 4.0 to 7.0. Thermodynamic properties of protein were estimated and at pH 7.0 the protein exhibited ΔG37of 26.31 kcal mole-1, ΔH of 204.27 kcal mole-1 and ΔS of 574.06 cal mole-1 K-1.Conclusion:The stability and thermodynamic analysis revealed that V. cholerae L-asparaginase was highly stable over a wide range of pH, with the highest stability in the pH range of 5.0–7.0.

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Isotope Effect and Kinetic Studies of the Reaction of Tertiary Alcohols with Triphenylphosphine–Carbon Tetrachloride: Ion Pair or Concerted?

Tetrahedron ◽

10.1016/s0040-4020(00)00277-5 ◽

2000 ◽

Vol 56 (22) ◽

pp. 3611-3617 ◽

Cited By ~ 7

Author(s):

Abduol Hossein Dabbagh ◽

Khalil Faghihi

Keyword(s):

Carbon Tetrachloride ◽

Isotope Effect ◽

Kinetic Studies ◽

Ion Pair ◽

Tertiary Alcohols

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Optical-optical double-resonance spectroscopy of the 1g(3P1) ion-pair state of Br2

Journal of Molecular Spectroscopy ◽

10.1016/0022-2852(88)90125-7 ◽

1988 ◽

Vol 127 (2) ◽

pp. 353-361 ◽

Cited By ~ 9

Author(s):

Takashi Ishiwata ◽

Kinichi Obi ◽

Ikuzo Tanaka

Keyword(s):

Double Resonance ◽

Ion Pair ◽

Resonance Spectroscopy ◽

Optical Double Resonance ◽

Double Resonance Spectroscopy ◽

Pair State

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General, Divergent Platform for Diastereoselective Synthesis of trans-Cyclooctenes with High Reactivity and Favorable Physiochemical Properties

10.26434/chemrxiv.13669160 ◽

2021 ◽

Author(s):

Jessica E. Pigga ◽

Julia Rosenberger ◽

Andrew Jemas ◽

Samantha Boyd ◽

Olga Dmitrenko ◽

...

Keyword(s):

Physicochemical Properties ◽

Large Scale ◽

High Reactivity ◽

Diels Alder ◽

Synthetic Methods ◽

High Yield ◽

Physiochemical Properties ◽

New Class ◽

State Models

trans-Cyclooctenes (TCOs) are essential partners for the fastest known bioorthogonal reactions, but current synthetic methods are limited by poor diastereoselectivity. Especially hard to access are hydrophilic TCOs with favorable physicochemical properties for live cell or in vivo experiments. Described is a new class of TCOs, ‘a-TCOs’, that is prepared in high yield via stereocontrolled 1,2-additions of nucleophiles to trans-cyclooct-4-enone, which itself was prepared on large scale in two steps from 1,5-cyclooctadiene. Computational transition state models rationalize the diastereoselectivity of 1,2-additions to deliver a-TCO products, which were also shown to be more reactive than standard TCOs and less hydrophobic than even a trans-oxocene analog. Illustrating the favorable physicochemical properties of a-TCOs, a fluorescent TAMRA derivative in live HeLa cells was shown to be cell-permeable through intracellular Diels-Alder chemistry and to washout more rapidly than other TCOs.

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