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Titolo:
MECHANISMS OF STABILIZATION OF THE INSULIN HEXAMER THROUGH ALLOSTERICLIGAND INTERACTIONS
Autore:
RAHUELCLERMONT S; FRENCH CA; KAARSHOLM NC; DUNN MF;
Indirizzi:
UNIV CALIF RIVERSIDE,DEPT BIOCHEM 015 RIVERSIDE CA 92521 UNIV CALIF RIVERSIDE,DEPT BIOCHEM 015 RIVERSIDE CA 92521 NOVO NORDISK AS,NOVO RES INST DK-2880 BAGSVAERD DENMARK
Titolo Testata:
Biochemistry
fascicolo: 19, volume: 36, anno: 1997,
pagine: 5837 - 5845
SICI:
0006-2960(1997)36:19<5837:MOSOTI>2.0.ZU;2-2
Fonte:
ISI
Lingua:
ENG
Soggetto:
HALF-SITE REACTIVITY; ZINC-BINDING DOMAINS; TRANSITION; PHENOL; TRANSFORMATION; COORDINATION; INTERMEDIATE; RESOLUTION; STABILITY; COMPLEX;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
42
Recensione:
Indirizzi per estratti:
Citazione:
S. Rahuelclermont et al., "MECHANISMS OF STABILIZATION OF THE INSULIN HEXAMER THROUGH ALLOSTERICLIGAND INTERACTIONS", Biochemistry, 36(19), 1997, pp. 5837-5845

Abstract

The insulin hexamer is an allosteric protein capable of undergoing transitions between three conformational states: T-6, T3R3, and R-6. These transitions are mediated by the binding of phenolic compounds to the R-state subunits, which provide positive homotropic effects, and by the coordination of anions to the bound metal ions, which act as heterotropic effecters. Since the insulin monomer is far more susceptible than the hexamer to thermal, mechanical, and chemical degradation, insulin-dependent diabetic patients rely on pharmaceutical preparations ofthe Zn-insulin hexamer, which act as stable forms of the biologicallyactive monomeric insulin. In this study, the chromophoric chelator 2,2',2 ''-terpyridine (terpy) has been used as a kinetic probe of insulin hexamer stability to measure the effect of homotropic and heterotropic effecters on the dissociation kinetics of the Zn2+- and Co2+-insulin hexamer complexes. We show that the reaction between terpy and the R-state-bound metal ion is limited by the T3R3 reversible arrow T-6 Or R-6 reversible arrow T3R3 conformational transition steps and the dissociation of one anionic ligand, or one anionic ligand and three phenolic ligand molecules, respectively, for T3R3 and R-6. Consequently, because the activation energies of these steps are dominated by the ground-state stabilization energy of the R-state species, the kinetic stabilization of the insulin hexamer toward terpy-induced dissociation is linked to the thermodynamic stabilization of the hexamer. The mass action effect of anion binding and, foremost, of phenolic ligand binding provides the major mechanism of stabilization, resulting in the tightening of the tertiary and quaternary hexamer structures. Using this kinetic method, we show that the R-6 conformation of Zn-insulin in the presence of Cl- ion and resorcinol is > 1.5 million-fold more stable thanthe T-3 units Of T-6 and T3R3 and >70 000-fold more stable than the R-3 unit of T3R3. Furthermore, the stabilization effect is correlated with the affinity of the ligands: the tighter the binding, the slower the reaction between terpy and R-state-bound metal ion. These concepts provide a new basis for the pharmaceutical improvement of the physicochemical stability of formulations both for native insulin and for fast-acting monomeric insulin analogues through ligand-mediated allostericinteractions.

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Documento generato il 01/10/20 alle ore 01:05:33