Conformational Adaptability in Enzymes

Citri, Nathan

doi:10.1002/9780470122822.ch7

Cited by 75 publications

(9 citation statements)

References 796 publications

(185 reference statements)

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“…The monophasic Michaelis–Menten relationship of the monomeric epimerase demonstrated the absence of such artefacts and the requirement for a dimeric structure to explain the regulation. The difference in V max values among different forms of the epimerase is a reflection of change of conformation (Table 1) [29,30]. The functional distinction between native and monomeric epimerase was also apparent from the inhibitory pattern with 5′‐UMP (Fig.…”

Section: Discussionmentioning

confidence: 99%

UDP‐galactose 4‐epimerase from Kluyveromyces fragilis – catalytic sites of the homodimeric enzyme are functional and regulated

Brahma¹,

Banerjee²,

Bhattacharyya³

2009

The FEBS Journal

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UDP‐galactose 4‐epimerase from Kluyveromyces fragilis is a homodimer containing one catalytic site and one NAD+ as cofactor per subunit. One 5′‐UMP, a competitive inhibitor, binds per dimer of epimerase as isolated and causes inactivation. Addition of 0.2 mm inhibitor to the enzyme in vitro leads to three sequential steps: first, the inhibitor binds to the unoccupied site; second, the inhibitor bound ex vivo is displaced allosterically; and finally, both sites are occupied by the inhibitor. These reactions have been monitored by kinetic lag in substrate conversion, coenzyme fluorescence, protection against trypsin digestion, and reductive inhibition. The transition profiles indicate the existence of a stable intermediate with one inhibitor‐binding site remaining unoccupied. Reductive inhibition of this intermediate reduced the activity to 58% ± 2%, with modification of one catalytic site. A change of conformation of the epimerase upon binding with substrate or inhibitor was evident from fluorescence emission spectra. The epimerase demonstrated a biphasic Michaelis–Menten dependency. The epimerase devoid of 5′‐UMP showed a Michaelis–Menten dependency that can be explained by assuming simultaneous operation of two catalytic sites. A monomeric form of the epimerase was devoid of such regulation. The inhibitory profile of 5′‐UMP also suggested negative cooperativity. Incubation of the epimerase with combinations of substrate analogs rendered one of the sites inactive, supporting the presence of two functional and regulated catalytic sites. Dissimilar kinetic patterns of the reconstituted enzyme after treatment with p‐chloromercuribenzoate indicated stability of the dimeric enzyme against fast association–dissociation, which could otherwise generate multiple forms of the enzyme with functional heterogeneity.

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Section: Discussionmentioning

confidence: 99%

UDP‐galactose 4‐epimerase from Kluyveromyces fragilis – catalytic sites of the homodimeric enzyme are functional and regulated

Brahma¹,

Banerjee²,

Bhattacharyya³

2009

The FEBS Journal

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“…Such conformational changes reduce enzyme flexibility, thereby enhancing stability. Citri (1973) reviewed the effect of ligands on enzyme thermostability and concluded that stabilization was prima-facie evidence that a conformational change had occurred.…”

Section: Sfructurul and Thermodynamic Considerutionsmentioning

confidence: 99%

Review: Enzyme inactivation during heat processing of food‐stuffs

Adams

1991

Int J of Food Sci Tech

133

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Enzyme inactivation during heat processing is reviewed with regard to fundamental aspects (structure, thermodynamics and kinetics), mathematical models and the relationship between enzyme activity and food quality. Enzyme stability is related to enzyme structure and to factors in the microenvironment. Kinetics of inactivation are categorized with respect to reaction order and two models are briefly discussed which describe the variation of inactivation rates with temperature. The determination of accurate kinetic parameters is emphasized where the objective is to develop mathematical models of enzyme inactivation in real foods. The value of modelling inactivation is assessed. Enzymes having effects on sensory quality are reviewed with particular emphasis on those enzymes wiiich influence flavour, through lipid degradation, and those which affect texture by catalysing the breakdown of starch, pectin or protein.

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“…H‐isotope exchange experiments 13, 14 maintained this concept until the advent of molecular dynamics simulations and high‐resolution proton NMR measurements on proteins. In an elegant review published in 1973, Citri 15 highlighted “the deep conceptual difference between a selective and an instructive role for the ligand”. Here, the concept of “fluctuation fit” proposed by Straub and Szabolcsi was summarized as an alternative theory, which explicitly postulates a ligand‐induced shift in the equilibrium of the enzyme, which fluctuates between various conformational states, while the substrate stabilizes the conformation which is optimal for binding and catalysis.…”

Section: Introductionmentioning

confidence: 99%