Implication of an Ionizing Group in the Control of Conformation and Activity of Chymotrypsin

Oppenheimer, Hannah L.; Labouesse, Bernard; Hess, George P.

doi:10.1016/s0021-9258(18)96598-1

Cited by 196 publications

(33 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…No uncatalysed reaction of the serine-195 anion was detected even at pH 10.5. The pKa of 9.5 is slightly higher than usually found presumably because of the presence of a small amount of organic solvent (Kaplan & Laidler, 1967) and may be attributed to the deprotonation of the a-amino group of isoleucine-16 (Oppenheimer, Labouesse & Hess, 1966) and its effect on substrate binding. The pKa of 7 obtained for both acylation and deacylation is more puzzling.…”

Section: Resultsmentioning

confidence: 78%

Catalytic activity of α-chymotrypsin in which histidine-57 has been methylated

Henderson

1971

Biochemical Journal

View full text Add to dashboard Cite

The properties of a derivative of alpha-chymotrypsin in which histidine-57 has been methylated have been examined. Although the modified enzyme binds substrate with the same affinity as does native alpha-chymotrypsin, acylation and deacylation occur at much decreased rates. As for native alpha-chymotrypsin, a basic group of pK(a) approx. 7 is involved in both acylation and deacylation. The significance of these results is considered in relation to the normal function of histidine-57.

show abstract

Section: Resultsmentioning

confidence: 78%

Catalytic activity of α-chymotrypsin in which histidine-57 has been methylated

Henderson

1971

Biochemical Journal

View full text Add to dashboard Cite

show abstract

“…8.5 associated with the conformational change that inactivates chymotrypsin 1989 8.67 5.27 856 I '3C-n.m.r. investigation of an inhibitor-a-chymotrypsin complex (Oppenheimer et al, 1966;Himoe et al, 1967;McConn et al, 1969;Ghelis et al, 1970;Fersht & Requena, 1971). The conformational change results from the ionization of the isoleucine-16/aspartate-194 ion-pair that inactivates chymotrypsin as the carboxy group of aspartate-194 moves and protrudes into the active site, preventing substrate binding (Sigler et al, 1968).…”

Section: Discussionmentioning

confidence: 99%

“…The binding of ligands such as indole raises the pKa of the isoleucine-16/aspartate-194 ion-pair to approx. 10.5 , whereas chemical modification of the hydroxy group of serine-195 by groups such as di-isopropyl phosphorofluoridate prevents the conformational change by raising the pKa of the ion-pair to > 11 (Oppenheimer et al, 1966;Sigler et al, 1968;McConn et al, 1969). Therefore we would expect Tos-Phe-CH2Cl-inhibited a-chymotrypsin to be locked in the 'active conformation' at least up to pH 11.…”

Section: Discussionmentioning

confidence: 99%

A 13C-n.m.r. investigation of the ionizations within an inhibitor–α-chymotrypsin complex. Evidence that both α-chymotrypsin and trypsin stabilize a hemiketal oxyanion by similar mechanisms

Finucane

Hudson

Malthouse

1989

Biochemical Journal

103

View full text Add to dashboard Cite

13C-n.m.r. was used to investigate the structure of the inhibitor enzyme complex formed when alpha-chymotrypsin is alkylated by L-1-chloro-4-phenyl-3-tosylamido-[2-13C]butan-2-one. Two signals are detected. The one at 204.82 +/- 0.11 p.p.m. does not titrate from pH 3 to 9 and is assigned to alkylated methionine-192. The second signal titrates from 99.08 p.p.m. to 103.44 p.p.m. with pKa 8.67. This signal is assigned to a tetrahedral adduct formed between the hydroxy group of serine-195 and the inhibitor. The titration shift of the tetrahedral adduct is ascribed to the ionization of the hemiketal hydroxy group. It is proposed that the resulting oxyanion is stabilized by interaction with the imidazolium ion of histidine-57. It is argued that this interaction must raise the pKa of at least 70% of histidine-57 to greater than 11. On denaturation/autolysis of the inhibitor-enzyme complex neither of the signals associated with the intact complex is detected, but a new signal is observed that titrates from 203.52 p.p.m. to 206.08 p.p.m. with pKa = 5.27. This titration shift is assigned to the ionization of the imidazolium ion of alkylated histidine, confirming that the inhibitor has alkylated histidine-57. The significance of these results for the catalytic mechanism of the serine proteinases is discussed.

show abstract

“…The free a-amino group of Ile 16 is generated by proteolytic cleavage between the EGF2 and protease domains, which converts VII to VIIa. The ability to form the salt bridge between the a-amino group of Ile 16 and the carboxyl side chain of Asp 194 is essential for stabilizing the active configuration of the catalytic triad (61). Based on these results, Higashi et al (8), proposed that when VIIa is in the unbound state, the heavy chain retains zymogen-like features, and steric constraints prevent formation of the critical salt bridge between Ile 16 and Asp 194 .…”

Section: Segmental Flexibility Conformational Change and Viia Activationmentioning

confidence: 99%

Loop Dynamics of the Extracellular Domain of Human Tissue Factor and Activation of Factor VIIa

Minazzo

Darlington

Ross

2009

Biophysical Journal

View full text Add to dashboard Cite

In the crystal structure of the complex between the soluble extracellular domain of tissue factor (sTF) and active-site-inhibited VIIa, residues 91 and 92 in the Pro(79)-Pro(92) loop of sTF interact with the catalytic domain of VIIa. It is not known, however, whether this loop has a role in allosteric activation of VIIa. Time-resolved fluorescence anisotropy measurements of probes covalently bound to sTF mutants E84C and T121C show that binding uninhibited Factor VIIa affects segmental motions in sTF. Glu(84) resides in the Pro(79)-Pro(92) loop, and Thr(121) resides in the turn between the first and second antiparallel beta-strands of the sTF subdomain that interacts with the Gla and EGF1 domains of VIIa; neither Glu(84) nor Thr(121) makes direct contact with VIIa. Probes bound to T121C report limited segmental flexibility in free sTF, which is lost after VIIa binding. Probes bound to E84C report substantial segmental flexibility in the Pro(79)-Pro(92) loop in free sTF, which is greatly reduced after VIIa binding. Thus, VIIa binding reduces dynamic motions in sTF. In particular, the decrease in the Pro(79)-Pro(92) loop motions indicates that loop entropy has a role in the thermodynamics of the protein-protein interactions involved in allosteric control of VIIa activation.

show abstract

Implication of an Ionizing Group in the Control of Conformation and Activity of Chymotrypsin

Cited by 196 publications

References 40 publications

Catalytic activity of α-chymotrypsin in which histidine-57 has been methylated

Catalytic activity of α-chymotrypsin in which histidine-57 has been methylated

A 13C-n.m.r. investigation of the ionizations within an inhibitor–α-chymotrypsin complex. Evidence that both α-chymotrypsin and trypsin stabilize a hemiketal oxyanion by similar mechanisms

Loop Dynamics of the Extracellular Domain of Human Tissue Factor and Activation of Factor VIIa

Contact Info

Product

Resources

About