The Structure of Papain

Drenth, Jan; Jansonius, Johan N.; Koekoek, Roelof; Wolthers, B.G.

doi:10.1016/s0065-3233(08)60279-x

Cited by 265 publications

(142 citation statements)

References 60 publications

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“…Therefore, pK, may only belong to the base which forms an ion-pair with the thiol group [3]. According to X-ray diffraction analysis the base interacting with the thiol group proved to be an imidazole group [26].…”

Section: Discussionmentioning

confidence: 99%

Ion‐Pair Formation as a Source of Enhanced Reactivity of the Essential Thiol Group of d‐Glyceraldehyde‐3‐Phosphate Dehydrogenase

Polgár

1975

European Journal of Biochemistry

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The reactivity and the mode of activation of the essential -SH group (Cys-149) of D-glyceraldehyde-3-phosphate dehydrogenase have been studied by means of a spectrophotometric method [Polgar, L., FEBS Lett. 38, 187-190 (1974)], capable of detecting the dissociated form of the thiol group in proteins.Alkylations of Cys-149 of NAD-free ~-glyceraldehyde-3-phosphate dehydrogenase with iodoacetamide and iodoacetate were investigated. The corrected absorbance change on alkylation at 250 nm (which is a direct parameter of the dissociation of the thiol group) and the alkylation rate were determined as a function of pH. The pH profiles of both dissociation and alkylation rate of Cys-149 conform to doubly sigmoid curves. All these curves implicate two ionizing groups (pK, = 5.5, pK2 = 8.2).It is concluded that there are two reactive forms of the -SH group in the apoenzyme between pH 5 and 10. One reactive form corresponds to the free mercaptide ion. The other can be identified with an ion-pair composed of a mercaptide ion and some base, possibly the imidazolium group of His-176.The ion-pair has lower molar absorption coefficient and nucleophilicity than the free mercaptide ion.The two reactive forms are transformed into each other with pK2 = 8.2. The ion-pair decomposes to a nondissociated thiol group and a protonated base with pK, = 5.5.In the presence of NAD, only the pH-rate profile of alkylation of ~-glyceraldehyde-3-phosphate dehydrogenase was measured (at 370 nm). Using iodoacetamide as alkylating agent we also obtained a doubly sigmoid curve. A slight downward shift on pK, and an upward shift in pK2 indicate that the ion-pair exists in a somewhat wider pH-range in the enzyme-coenzyme complex.An increase in the ionic strength of the reaction mixture from 0.09 to 0.45 M does not abolish the doubly sigmoid character of the curves determined either in the presence or in the absence of NAD.Previous studies have shown that the -SH group of thiolsubtilisin [1,2] and papain [3] forms an ionpair with the imidazole group of a neighboring histidine residue. At slightly acidic pH this mercaptideimidazolium ion-pair may account for the high reactivity as compared to what is expected from the normal dissociation of an -SH group. Thus the pHdependence of alkylation of papain with haloacetamides displays a doubly sigmoid curve with parameters corresponding to the reactivity of the free Enzyme. D-Glyceraldehyde 3-phosphate : NAD oxidoreductase (phosphorylating) (EC 1.2

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Section: Discussionmentioning

confidence: 99%

Ion‐Pair Formation as a Source of Enhanced Reactivity of the Essential Thiol Group of d‐Glyceraldehyde‐3‐Phosphate Dehydrogenase

Polgár

1975

European Journal of Biochemistry

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“…Thiolsubtilisin can be regarded as a model of thiol proteases, such as papain, inasmuch as both thiolsubtilisin [2] and papain [3] have an imidazole ring in the immediate vicinity of the thiol group, as clearly indicated by X-ray diffraction studies. However, thiolsubtilisin is not a protease, it can only catalyze the hydrolysis of active esters [a].…”

Section: By Alkylating Thiolsubtilisin With Enantiomeric Reactants Amentioning

confidence: 99%

On the Reactivity of the Thiol Group of Thiolsubtilisin

Polgár

Halász

Moravcsik

1973

European Journal of Biochemistry

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1. The reaction of thiolsubtilisin with iodoacetamide shows that in the alkaline pH-range the -SH group ofthe enzyme reacts like a simple thiol compound such as mercaptoacetate. On the other hand, around neutral pH the enzyme displays enhanced reactivity compared with that of mercaptoacetate. On the basis of measurements in aHaO it is concluded that a mercaptideimidazolium ion-pair is formed in thiolsubtilisin and this accounts for the enhanced reactivity around neutral pH.2. Iodoacetamide reacts with the -SH group of the enzyme, as compared to mercaptoacetate, at a relatively higher rate than bromo-, chloro-, and fluoroacetamide. This might be due to the less polar environment around the -SH group on the surface of the enzyme than in water, as indicated by alkylation of mercaptoacetate with iodo-and chloroacetamide in the presence of dioxane.3. By alkylating thiolsubtilisin with enantiomeric reactants, a slight conformational change at the active site, not measurable with ordinary physical methods, could be established above pH z 9, whereas the overall protein structure is stable up to pH zz 11. D-2-Bromo-n-butyramide reacts with thiolsubtilisin about 60 times as fast as D-2-bromopropionamide. This indicates that hydrophobic interaction between the enzyme and a methyl group of the substrate can enhance the reaction rate by more than one order of magnitude. Model building of the active site suggests that Ala-152 and Thr-220 of thiolsubtilisin may form van der Waals interactions with D-2-bromon-butyramide if the alkylating agent is properly positioned for reaction. No such interaction is possible between D-2-bromopropionamide and thiolsubtilisin.Thiolsubtilisin, an -SH analog of the serine protease subtilbin, can be produced by the replacement of the reactive serine residue at the active site by a cysteine residue [l]. Thiolsubtilisin can be regarded as a model of thiol proteases, such as papain, inasmuch as both thiolsubtilisin [2] and papain [3] have an imidazole ring in the immediate vicinity of the thiol group, as clearly indicated by X-ray diffraction studies. However, thiolsubtilisin is not a protease, it can only catalyze the hydrolysis of active esters [a].In a recent paper we have pointed out that the catalytic action of papain depends on the formation of a mercaptide-imidazolium ion-pair at the active site [5]. On the basis of acylation of thiolsubtilisin, we have also suggested that the reactivity of the thiol group is highly affected by an interaction with the The detailed study of alkylation of Carlsberg thiolsubtilisin is presented in this paper. Unlike acylation, alkylation is a simple nucleophilic displacement reaction, not complicated by the formation of an intermediate, therefore the study of alkylation, rather than of acylation, offers more conclusive evidence for the chemical behavior of the thiol group. Carlsberg thiolsubtilisin was preferred to the Novo enzyme because of its higher stability. We have found that around neutral pH values there is indeed an interaction between the thiol and imi...

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“…Trypsin, Cat-H and PV 3Cpro belong to 3 distinct families of proteases; upon comparing the sequences of other members of these families, we did not find the same principal catalytic residues (Ser or Cys), a reasonable similarity was observed ( fig.3) ; unpublished), the aligned segments represent the most highly conserved region of the molecules. In cellular serine and cysteine proteinases, these segments form topologically distinct loops [43,44]. The tertiary structure of the viral proteinases discussed here is unknown, but theoretical predictions of their secondary structure suggest that the region in question forms a &fold similar to the serine loop of chymotrypsin-like proteinases.…”

Section: Resultsmentioning

confidence: 92%

Poliovirus‐encoded proteinase 3C: a possible evolutionary link between cellular serine and cysteine proteinase families

1986

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Here we demonstrate significant similarities between the amino acid sequences of trypsin (a serine protease) and the N-terminal piece of a specific fragment of the poliovirus polyprotein encompassing the sequence of the viral proteinase 3C, and also between cathepsin H (a cysteine protease) and the C-terminal piece of the same fragment. A coherent alignment of the sequences of the 3 proteases was obtained, in which the principal catalytically active residues occupy identical positions. A hypothesis is proposed that the viral enzyme may provide an evolutionary link between serine and cysteine protease families.

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The Structure of Papain

Cited by 265 publications

References 60 publications

Ion‐Pair Formation as a Source of Enhanced Reactivity of the Essential Thiol Group of d‐Glyceraldehyde‐3‐Phosphate Dehydrogenase

Ion‐Pair Formation as a Source of Enhanced Reactivity of the Essential Thiol Group of d‐Glyceraldehyde‐3‐Phosphate Dehydrogenase

On the Reactivity of the Thiol Group of Thiolsubtilisin

Poliovirus‐encoded proteinase 3C: a possible evolutionary link between cellular serine and cysteine proteinase families

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