Studies of the Chymotrypsinogen Family. V. The Effect of Small-Molecule Contaminants on the Kinetic Behavior of α-Chymotrypsin<sup>1</sup>

Yapel, Anthony F.; Han, Moon Hi; Lumry, Rufus; Rosenberg, Andreas; Shiao, Da Fong

doi:10.1021/ja00963a036

Cited by 60 publications

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“…The decrease of the pKs of exposed tyrosines fits also with the conformational changes that chymotrypsinogen is known to undergo upon activation [41-431. The finding of a single pK value for the tyrosine residues of both chymotrypsin and chymotrypsinogen by Marini and Wunsch [5] is in disagreement with all other data, and could be partially explained by the presence of impurities of peptide type in their protein sample [44,45]. I n our study it is most likely that ammonium sulfate fractionation, chromatography on CM-Sephadex, and extensive dialysis have removed any trace of the contaminants present in the commercial product.…”

Section: Discussionmentioning

confidence: 99%

Study of the Groups Controlling the Activity and Conformation of Chymotrypsin at Alkaline pH: N‐Terminal Isoleucine and Tyrosines

Karibian¹,

Laurent²,

Labouesse³

et al. 1968

European Journal of Biochemistry

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It is shown that the change in activity and structure of acetyl-6-chymotrypsin in the pH 7-11 range is not correlated with the ionizations of tyrosine residues.The pKs of the two titratable tyrosines of acetyl-8-chymotrypsin are found to be 10.2 and 11.5, a t 15' (9.6 and 10.8 after correction for electrostatic interactions), and those of the starting acetylated zymogen are 10.5 and 12.1 (10.0 and 11.5 after identical corrections). The plot of log rate of deacetylation of the titratable tyrosines versus pH is linear between pH 9 and pH 12. At a given pH there is a linear relation between the log deacetylation rate and the pKs of the residues. The pH rate profile of acylation of acetyl-8-chymotrypsin by 4-carboxy-3-nitrophenyl-N,N-diphenylcarbamate is bell-shaped, with a maximum a t pH 7.9 a t 15", in 0.14 M KC1. The right side of the curve shows a pKapp of 8.9, whether the exposed titrable tyrosines of the enzyme are acetylated or not. A similar pKapp (9.1) is found for the pH-dependent change in optical rotation of the acetylated enzyme, when the exposed tyrosines are acetylated. After correction for electrostatic interactions, this apparent pK becomes 8.4 and can be assigned only to the a-aminogroup of the N-terminal isoleucine.Recently, conflicting reports have been presented on the role [l --31 of the two exposed tyrosine residues in chymotrypsin, and on their ionization state [4-71. Values of pK ranging from 9.1 [4,7] to 10.6 [5] have been assigned to them. Considerable evidence has shown that a n ionizable group of p K about 9 is involved in the control of activity [8-111 and structure [12-151 of chymotrypsin. With this evidence and the X-ray work of Matthews et al. [16], it seemed useful to examine how tyrosine ionization is involved in the active conformation of the protein and how it interferes with that of the N-terminal isoleucine residue of the B chain of the enzyme. The latter amino acid has emerged recently as the most probable residue controlling chymotrypsin activity a t alkalineThe work reported here was carried out using acetylated 8-chymotrypsin (Ac-CT), made by activation of acetylated chymotrypsinogen (Ac-CTG) pH [17-191. Non-Standard Abbreviations. Ac-Tyr-OEt, N-acetyl-Ltyrosine ethylester ; DPCC diphenylcarbamyl chloride; NCDC, 4-carboxy-3nitrophenyl-N,N-diphenylcarbamate; Ac-Tyr-NH,, N-acetyl-L-tyrosinamide; Ac,-Tyr-NH,, N,Odiacetyl-L-tyrosinamide ; Ac-CTG, refers to chymotrypsinogen with all of its s-NH, groups of lysine and its a-NH, group of half-cystine acetylated; Ac-CT, refers to S-chymotrypsin obtained from Ac-CTG. When specified -Tyr(H) refers to proteins whose exposed tyrosines are free to ionize ; -Tyr(Ac) refers to proteins whose exposed tyrosines are acetylated.Enzymes. Chymotrypsin (EC 3.4.4.5) ; trypsin (EC 3.4.4.4).[17], so that the results would not be obscured by lysine or N-terminal half-cystine ionizations. Previous results [17] have shown that the €-amino groups of lysine residues and the a-amino group of N-terminal half-cystine are not related to the decrease in ...

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Section: Discussionmentioning

confidence: 99%

Study of the Groups Controlling the Activity and Conformation of Chymotrypsin at Alkaline pH: N‐Terminal Isoleucine and Tyrosines

Karibian¹,

Laurent²,

Labouesse³

et al. 1968

European Journal of Biochemistry

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“…As an aid to understanding the nature of the conformational changes mentioned above and also the various forces involved in the binding reaction, a knowledge of the enthalpic and entropic contributions to the free energies of binding are important. Several workers have estimated the enthalpies of binding of certain inhibitors and virtual substrates to a-CT from the temperature dependence of the binding constants (Doherty and Vaslow, 1952;Yapel, 1967;Hymes et al, 1969). Direct determination of these quantities by calorimetry has been reported by Canady and Laidler (1958) who studied the binding of hydrocinnamate ion to a-CT as a function of pH.…”

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confidence: 99%

Calorimetric investigations of the binding of inhibitors to α-chymotrypsin. I. Enthalpy of dilution of α-chymotrypsin and of proflavine, and the enthalpy of binding of indole, N-acetyl-D-tryptophan, and proflavine to α-chymotrypsin

Shiao¹,

Sturtevant²

1969

Biochemistry

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A flow microcalorimeter has been employed to measure the enthalpies of binding of indole, N-acetybtryptophan, and proflavin to a-chymotrypsin at pH 7.8 and also the heats of dilution of a-chymotrypsin and proflavin at the same pH. The following points are discussed. (1) The studies of the heats of dilution of a-chymotrypsin as a function of enzyme concentration lead to the suggestion that monomer-dimer equilibrium of this enzyme exists at pH 7.8. The calculated thermodynamic functions for this equilibrium are consistent with the view that the dimeric forms of the enzyme can be identified as the enzyme-substrate intermediates preceding the autolysis reaction. (2) The heat effects due to the dilution of proflavin are shown to be consistent with the assumption that the self-association of this compound can be T he binding of various inhibitors to a-CT1 has been extensively investigated by various techniques. Niemann and his colleagues examined over 100 compounds, determining their inhibition constants toward the CT-catalyzed hydrolysis of acetyl-L-valine (Hein and Niemann, 1962;Wallace et al., 1963). From those studies, useful information concerning the topography of the active site of a-CT was obtained. The comparison of binding constants of some charged and uncharged inhibitors as a function of pH (Foster and Niemann, 1955;Johnson and Knowles, 1966) has led to the suggestion that the active site of a-CT is negatively charged at neutral pH values where the molecule is enzymically active. This suggestion has recently received support from X-ray studies of a-CT (Blow et al., 1969).The binding of various substrates and inhibitors to a-CT produces an enhancement of the fluorescence of the enzyme (Sturtevant, 1962). The fluorescence increase takes place with first-order rate constants of about 1 sec-1 for widely different ligands, the rate constants being independent of ligand concentrations. These findings suggest that conformational changes in a-CT, which are included by the binding of substrates and inhibitors, are responsible for the observed * From the adequately represented by a series of equilibria involving dimer, trimer, and other higher polymers. The large negative enthalpy of polymerization (-4.0 kcal/mole) shows that the self-association of proflavin is accompanied by an unfavorable entropy change.(3) The apparent heats of binding of inhibitors to a-chymotrypsin were observed to be strongly dependent on enzyme concentration. These observations lead to the suggestion that the dimeric forms of the enzyme are incapable of binding inhibitors. (4) The enthalpies of binding of indole, N-acetyl-D-tryptophan, and proflavin to a-chymotrypsin were observed to be -15.2, -19.0, and -11.3 kcal/ mole, respectively, at pH 7.8. On the basis of these numbers it is concluded that conformational changes in the enzyme are induced by the binding of inhibitors. changes in fluorescence emission. As an aid to understanding the nature of the conformational changes mentioned above and also the various forces involved in the binding r...

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“…From Fig.3 it is clear that the anomalous effect of enzyme concentration on kobs is due to an impurity in commercial samples of a-chymotrypsin (cf. [18,19]) Further, the product of the reaction of the impurity with the acyl-enzyme is readily separated from the enzyme.…”

Section: Discussionmentioning

confidence: 99%

A Kinetic Study of the Deacylation of alpha-Benzamido-trans-cinnamoyl-chymotrypsin. Evidence for the Intervention of Non-Enzymic Species

et al. 1974

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The apparent first-order rate constant (kobs) for the deacylation of a-benzamido-transcinnamoyl-chymotrypsin, determined by direct observation at 310 nm, pH 7.9 (kobs = 0.102 s-l; K. Brockelhurst and K. Williamson 119671 Chem. Commun., 666), differs from kcat determined under the same conditions with [S], > [El, (kcat = 0.033 9-l). This result is contrary to other evidence indicating that kcat is a measure of the deacylation rate constant. The discrepancy has been shown to be due to the presence in commercial samples of a-chymotrypsin of impurities which catalyse the deacylation reaction under the conditions used for the determination of kobs ([El, > [S],). The impurities, which may be removed from enzyme solutions by gel filtration on Sephadex G-25 or G-50, have been shown to be autolysis products (presumably peptides) of or-chymotrypsin. Catalysis of deacylation results from attack of the free or-amino groups of these peptides on the acyl-enzyme, to give N-or-benzamido-trans-cinnamoyl-peptides. The consequences of these findings must be considered in interpreting any experiment in which deacylation is observed directly a t high enzyme concentration, with [El, > [S],. Particularly susceptible to error are experiments designed to determine the effect of pH on the deacylation rate constant, and literature results of some such experiments are critically assessed.Oxazolinones were originally introduced into enzymology as activated derivatives of N-acylamino acids to simplify the preparation of acyl-enzymes and to allow a simple determination of deacylation rate constants [l]. The reaction of 4-trans-benzylidene -2 -phenyloxazolin -5 -one (I) with a-chymo-"' \Ph was initially studied to allow the determination of the deacylation rate constant for a-benzamido-transcinnamoyl-chymotrypsin [2,3]. The a-benzamidocinnamoyl group contains the /3-aromatic substituent and the a-acylamino group characteristic of good substrates for a-chymotrypsin [4], but the spatial arrangement of these groups with respect to the serine ester bond in the acyl-enzyme is restricted by the double bond. However, conflicting results were obtained by Brocklehurst and Williamson [5] when the "deacylation" reaction was observed directly by the addition of trans-benzylidene-2-phenyloxazolin-5-one to an excess of enzyme, the reaction being followed by a decrease in absorbance a t 310 nm. The apparent firstorder rate constant, kobs, was found to show 8 sigmoidal pH-rate profile with k = 0.154 5-l and pKa' = 7.64. Thus, a t pH 7.93, the calculated value of kobs is 0.102 s-l, and since both kcat and kobs are purported to equal k+3, a factor of 3 exists between

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Studies of the Chymotrypsinogen Family. V. The Effect of Small-Molecule Contaminants on the Kinetic Behavior of α-Chymotrypsin¹

Cited by 60 publications

References 0 publications

Study of the Groups Controlling the Activity and Conformation of Chymotrypsin at Alkaline pH: N‐Terminal Isoleucine and Tyrosines

Study of the Groups Controlling the Activity and Conformation of Chymotrypsin at Alkaline pH: N‐Terminal Isoleucine and Tyrosines

Calorimetric investigations of the binding of inhibitors to α-chymotrypsin. I. Enthalpy of dilution of α-chymotrypsin and of proflavine, and the enthalpy of binding of indole, N-acetyl-D-tryptophan, and proflavine to α-chymotrypsin

A Kinetic Study of the Deacylation of alpha-Benzamido-trans-cinnamoyl-chymotrypsin. Evidence for the Intervention of Non-Enzymic Species

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Studies of the Chymotrypsinogen Family. V. The Effect of Small-Molecule Contaminants on the Kinetic Behavior of α-Chymotrypsin1

Cited by 60 publications

References 0 publications

Study of the Groups Controlling the Activity and Conformation of Chymotrypsin at Alkaline pH: N‐Terminal Isoleucine and Tyrosines

Study of the Groups Controlling the Activity and Conformation of Chymotrypsin at Alkaline pH: N‐Terminal Isoleucine and Tyrosines

Calorimetric investigations of the binding of inhibitors to α-chymotrypsin. I. Enthalpy of dilution of α-chymotrypsin and of proflavine, and the enthalpy of binding of indole, N-acetyl-D-tryptophan, and proflavine to α-chymotrypsin

A Kinetic Study of the Deacylation of alpha-Benzamido-trans-cinnamoyl-chymotrypsin. Evidence for the Intervention of Non-Enzymic Species

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Studies of the Chymotrypsinogen Family. V. The Effect of Small-Molecule Contaminants on the Kinetic Behavior of α-Chymotrypsin¹