The Influence of Mechanism on the Apparent pK<sub>a</sub>' of Participating Groups in Enzymic Reactions

Bruice, Thomas C.; Schmir, Gaston L.

doi:10.1021/ja01526a028

Cited by 54 publications

(38 citation statements)

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“…Despite the fact that these two figures are quite different, it can be considered that the activity curve hides a complex set of kinetic steps whose effect is a shift of the midpoint of the curve [53], and that it is an oversimplification to conclude that activity is not modified by tyrosine ionization. However, Fig.…”

Section: Relationship Between Tyrosine Ionization Andmentioning

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: Relationship Between Tyrosine Ionization Andmentioning

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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“…Widely recognized aspects of these problems include (1) perturbations of kinetically determined PKa values by general microenvironmental solvation effects (see, e.g., Schmidt & Westheimer, 1971), by specific interactions between proton-binding sites (see, e.g., H. Dixon & Tipton, 1973;Brocklehurst, 1974;Polgar, 1974;Shipton et al, 1975) and by pre-equilibria (Bruice & Schmir, 1959;Caplow, 1969) Vol. 155…”

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confidence: 99%

PH-dependence of the steady-state rate of a two-step enzymic reaction

Brocklehurst

Dixon

1976

Biochemical Journal

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1. The pH-dependence is considered of a reaction between E and S that proceeds through an intermediate ES under "Briggs-Haldane' conditions, i.e. there is a steady state in ES and [S]o greater than [E]T, where [S]o is the initial concentration of S and [E]T is the total concentration of all forms of E. Reactants and intermediates are assumed to interconvert in three protonic states (E equilibrium ES; EH equilibrium EHS; EH2 equilibrium EH2S), but only EHS provides products by an irreversible reaction whose rate constant is kcat. Protonations are assumed to be so fast that they are all at equilibrium. 2. The rate equation for this model is shown to be v = d[P]/dt = (kcat.[E]T[S]o/A)/[(KmBC/DA) + [S]o], where Km is the usual assembly of rate constants around EHS and A-D are functions of the form (1 + [H]/K1 + K2/[H]), in which K1 and K2 are: in A, the molecular ionization constants of ES; in B, the analogous constants of E; in C and D, apparent ionization constants composed of molecular ionization constants (of E or ES) and assemblies of rate constants. 3. As in earlier treatments of this type of reaction which involve either the assumption that the reactants and intermediate are in equilibrium or the assumption of Peller & Alberty [(1959) J. Am. Chem. Soc. 81, 5907-5914] that only EH and EHS interconvert directly, the pH-dependence of kcat. is determined only by A. 4. The pH-dependence of Km is determined in general by B-C/A-D, but when reactants and intermediate are in equilibrium, C identical to D and this expression simplifies to B/A. 5. The pH-dependence of kcat./Km, i.e. of the rate when [S]o less than Km, is not necessarily a simple bell-shaped curve characterized only by the ionization constants of B, but is a complex curve characterized by D/B-C. 6. Various situations are discussed in which the pH-dependence of kcat./Km is determined by assemblies simpler than D/B-C. The special situation in which a kcat./Km-pH profile provides the molecular pKa values of the intermediate ES complex is delineated.

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“…Any direct interaction of the carboxyl group with the metal ion should stabilize the carboxylate anion and therefore reduce the pK a . Likewise, a very favorable equilibrium prior to the ratelimiting step would reduce the apparent pK a (39). The high pK app values for III could reflect either a possible hydrogen bonding interaction of the carboxyl group with metal ion-bound water that stabilizes the proton on the carboxyl oxygen, or proximity of the carboxyl to the negative charge of the partially ionized metal ion bound water.…”

Section: Discussionmentioning

confidence: 99%

Metal Ion Catalysis in the Hydrolysis of Esters of 2-Hydroxy-1,10-phenanthroline: The Effects of Metal Ions on Intramolecular Carboxyl Group Participation

Fife

Pujari

2000

Bioorganic Chemistry

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The Influence of Mechanism on the Apparent pK_a' of Participating Groups in Enzymic Reactions

Cited by 54 publications

References 1 publication

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

PH-dependence of the steady-state rate of a two-step enzymic reaction

Metal Ion Catalysis in the Hydrolysis of Esters of 2-Hydroxy-1,10-phenanthroline: The Effects of Metal Ions on Intramolecular Carboxyl Group Participation

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The Influence of Mechanism on the Apparent pKa' of Participating Groups in Enzymic Reactions

Cited by 54 publications

References 1 publication

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

PH-dependence of the steady-state rate of a two-step enzymic reaction

Metal Ion Catalysis in the Hydrolysis of Esters of 2-Hydroxy-1,10-phenanthroline: The Effects of Metal Ions on Intramolecular Carboxyl Group Participation

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The Influence of Mechanism on the Apparent pK_a' of Participating Groups in Enzymic Reactions