Half-Site Reactivit

Seydoux, François; Malhotra, O.P.; Bernhard, Sidney A.

doi:10.3109/10409237409105448

Cited by 147 publications

(92 citation statements)

References 99 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The maintenance of the hexameric form there, despite the lack of the entire dimer ␤-strand interface, is somehow compatible with the association of DOI into a R 6 hexamer observed in the presence of Zn 2ϩ and cyclohexanol (29). Although the T 6 3 T 3 R 3 3 R 6 dynamic transitions are rather well described by the SMB model (51)(52)(53)(54)(55)(56), the conformational events on the monomer 3 dimer 3 hexamer pathway are much less understood. Our results presented here provide further evidence that the nature of the changes on the insulin dimer interfaces (and associated other parts of the insulin molecule) is quite asymmetrical (Fig.…”

Section: Impact Of Modifications On Binding Affinity Of Analogues-supporting

confidence: 69%

Non-equivalent Role of Inter- and Intramolecular Hydrogen Bonds in the Insulin Dimer Interface

Antolíková

Žáková

Turkenburg

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

Apart from its role in insulin receptor (IR) activation, the C terminus of the B-chain of insulin is also responsible for the formation of insulin dimers. The dimerization of insulin plays an important role in the endogenous delivery of the hormone and in the administration of insulin to patients. Here, we investigated insulin analogues with selective N-methylations of peptide bond amides at positions B24, B25, or B26 to delineate their structural and functional contribution to the dimer interface. All N-methylated analogues showed impaired binding affinities to IR, which suggests a direct IR-interacting role for the respective amide hydrogens. The dimerization capabilities of analogues were investigated by isothermal microcalorimetry. Insulin is an important polypeptide hormone that controls a wide range of cellular processes such as the regulation of blood glucose uptake and has a large impact on protein and lipid metabolism. However, despite decades of intensive research, many questions about the structure of insulin and its mechanism of action remain. The solid state-based structural insight into the insulin molecule is limited to inactive dimeric or hexameric storage forms (1-3), whereas the insulin monomer represents the active form of the hormone when binding to the insulin receptor (IR).3 It is also widely accepted that insulin undergoes a profound structural change during this process (4 -6), a hypothesis supported by a plethora of highly dynamic hormone conformers identified by NMR studies (7-13). Attempts to determine the structure of the insulin-IR complex have been unsuccessful so far. However, the regions of the insulin molecule responsible for the interaction with the IR (3, 14) or for its dimerization and hexamerization (15, 16) have been functionally and structurally identified in a number of insulin analogues.The insulin molecule consists of two peptide chains, a 21-amino acid A-chain and a 30-amino acid B-chain, interconnected by two interchain and one intrachain disulfide bridges. The C terminus of the B-chain of insulin, particularly residues B24 -B26, plays a substantial role in the initial contact with the receptor. It is believed that the C terminus of the B-chain of insulin must be detached away from the central B-chain ␣-helix of insulin (2, 6). One of the main signatures of this so-called "active form" of insulin should be the exposure of the previously hidden amino acids Gly-A1, Ile-A2, and Val-A3, which are important for the interaction with IR (3). Recently, we described crystal structures of several shortened and full-length insulin analogues with modifications at the B26 position (17). The structural convergence of some of these highly active analogues (200 -400%) enabled us to postulate that the active form of human insulin is characterized by a formation of a new type II ␤-turn at positions B24 -B26.Besides its role in IR activation and IR negative cooperativity (18,19), the C terminus of the B-chain is also responsible for the formation and stabilization of the insulin dimers t...

show abstract

Section: Impact Of Modifications On Binding Affinity Of Analogues-supporting

confidence: 69%

Non-equivalent Role of Inter- and Intramolecular Hydrogen Bonds in the Insulin Dimer Interface

Antolíková

Žáková

Turkenburg

et al. 2011

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…It has been shown previously that the four 3-phosphoglyceroyl groups of the fully acylated tetramer were not kinetically equivalent in various specific reactions [33]. The present results show that this non-equivalence among acylated sites cannot be explained by a different extent of saturation of these sites by NAD+.…”

Section: Coenzyme-binding Properties Of Acyluted Glyceraldehqde-3-phcontrasting

confidence: 44%

“…Hence, the introduction of the 3-phosphoglyceroyl group in the vicinity of the nicotinamidebinding subsite [31] reduces considerably the binding affinity of the coenzyme and modifies significantly the interprotomeric interactions which are responsible for the anticooperative properties of the unacylated enzyme. In view of recent crystallographic evidence [32] for pairwise structural asymmetry in the lobster holo( NAD + )enzyme, two distinct interpretations of the present results can be proposed: (a) the preexistent asymmetry of the apoenzyme is destroyed upon the 'all-of-the-sites' acylation of the enzyme tetramer [33,34]; (b) the acylation of the enzyme modifies the tertiary and quaternary structures of the enzyme in such a way that the transmission of negative hoinotropic effects [35] through the R axis relating the two subunits of the functional dimer is no longer operating upon NAD' binding. Irrespective of the preferred alternative, it appears that a specific 'all-ofthe-sites' modification of the enzyme tetramer does affect the functional properties supported by the quaternary structure of the enzyme.…”

Section: Coenzyme-binding Properties Of Acyluted Glyceraldehqde-3-phmentioning

confidence: 57%

Specific Interactions of 3-Phosphoglyceroyl-glyceraldehyde-3-Phosphate Dehydrogenase with Coenzymes

et al. 1976

View full text Add to dashboard Cite

The binding of oxidized and reduced coenzyme (NAD ' and NADH) to 3-phosphoglyceroylglyceraldehyde-3-phosphate dehydrogenase has been studied spectrophotometrically and fluorimetrically. The binding of NAD+ to the acylated sturgeon enzyme is characterized by a significant quenching of the enzyme fluorescence (about 25 '%;) and the induction of a difference spectrum in the ultraviolet absorbance region of the enzyme. Both of these spectroscopic properties are quantitatively distinguishable from those of the corresponding binary enzyme . NAD ' complex. Binding isotherms estimated by gel filtration of the acylated enzyme are in close agreement to those obtained by spectrophotometric and fluorimetric titrations. Up to four NAD' molecules are bound to the enzyme tetramer. No anticooperativity can be detected in the binding of oxidized coenzyme, which is well described on the basis of a single class of four binding sites with a dissociation constant of 25 pM at 10 "C, pH 7.0.The binding of NADH to the acylenzyme has been characterized spectrophotometrically. The absorption band of the dihydronicotinamide moiety of the coenzyme is blue-shifted to 335 nm with respect to free NADH. In addition, a large hypochromicity (23%) is observed together with a significant increase of the bandwidth at half height of this absorption band. This last property is specific to the acylenzyme . NADH complex, since it disappears upon arsenolysis of the acylenzyme.The binding affinity of NADH to the acylated enzyme has been estimated by performing simultaneous spectrophotometric and fluorimetric titrations of the NADH appearance upon addition of NAD' to a mixture of enzyme and excess glyceraldehyde 3-phosphate. In contrast to NAD', the reduced COenzyme NADH appears to be relatively strongly bound to the acylated enzyme, the dissociation constant of the acylenzyme . NADH complex being estimated as 2.0 pM at 25 "C. In addition a large quenching of the NADH fluorescence (about 83 yo) is observed. The comparison of the dissociation constants of the coenzyme . acylenzyme complexes and the corresponding Michaelis constants suggests a reaction mechanism of the enzyme in which significant formation and dissociation of NAD' . acylenzyme and NADH . acylenzyme complexes occur. Under physiological conditions the activity of the enzyme can be regulated by the ratio of oxidized and reduced coenzymes.Possible reasons for the lack of anticooperativity in coenzyme binding to the acylated form of the enzyme are discussed.

show abstract

“…E allo -Native-A/E cat -Native-B and E allo -Native-B/E catNative-A) that do not readily interchange. This would put native PGHS-2 into the category of pre-existent stable conformational heterodimers of which there now are numerous examples (55,56,60,61). Recent NMR studies have challenged the idea that there are structural differences in enzymes such as tyrosyl-tRNA synthetase, tryptophanyl-tRNA synthetase, and cAMP receptor protein (61); however, there is functional evidence that these three enzymes are pre-existent conformational heterodimers.…”

Section: Pghs-2 As a Pre-existent Conformational Heterodimer-mentioning

confidence: 99%

Pre-existent Asymmetry in the Human Cyclooxygenase-2 Sequence Homodimer

Sharma

Jurban

Smith

2013

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: Cyclooxygenase-2 (COX-2), a target of coxibs, aspirin, and related drugs, is a sequence homodimer that functions as a conformational heterodimer. Results: Kinetic and aspirin labeling studies indicate that COX-2 is composed of two equivalent, stable populations of conformational heterodimers. Conclusion: COX-2 is processed and folds into a pre-existent conformational heterodimer. Significance: COX-2 half-site functionality results from COX-2 folding into a stable conformational heterodimer.

show abstract

Half-Site Reactivit

Cited by 147 publications

References 99 publications

Non-equivalent Role of Inter- and Intramolecular Hydrogen Bonds in the Insulin Dimer Interface

Non-equivalent Role of Inter- and Intramolecular Hydrogen Bonds in the Insulin Dimer Interface

Specific Interactions of 3-Phosphoglyceroyl-glyceraldehyde-3-Phosphate Dehydrogenase with Coenzymes

Pre-existent Asymmetry in the Human Cyclooxygenase-2 Sequence Homodimer

Contact Info

Product

Resources

About