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Cation induced differential effect on structural and functional properties of Mycobacterium tuberculosis alpha-isopropylmalate synthase.

Singh K, Bhakuni V - BMC Struct. Biol. (2007)

Bottom Line: The activator K+ also interacts with the catalytic TIM barrel domain however, it does not induce any significant effect on the enzyme structure.This divalent cation induced structural cooperativity might result in modulation of activity of the catalytic domain by regulatory domain.The studies for the first time demonstrate that different cations bind at different sites in the enzyme leading to their differential effects on the structure and functional activity of the enzyme.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow, India. ksinghcdri@gmail.com <ksinghcdri@gmail.com>

ABSTRACT

Background: Alpha-isopropylmalate synthase (MtalphaIPMS), an enzyme that catalyzes the first committed step of the leucine biosynthetic pathway of Mycobacterium tuberculosis is a potential drug target for the anti-tuberculosis drugs. Cations induce differential effect of activation and inhibition of MtalphaIPMS. To date no concrete mechanism for such an opposite effect of similarly charged cations on the functional activity of enzyme has been presented.

Results: Effect of cations on the structure and function of the MtalphaIPMS has been studied in detail. The studies for the first time demonstrate that different cations interact specifically at different sites in the enzyme and modulate the enzyme structure differentially. The inhibitors Zn2+ and Cd2+ ions interact directly with the catalytic domain of the enzyme and induce unfolding/denaturation of the domain. The activator K+ also interacts with the catalytic TIM barrel domain however, it does not induce any significant effect on the enzyme structure. Studies with isolated catalytic TIM barrel domain showed that it can carry out the catalytic function on its own but probably requires the non-catalytic C-terminal domain for optimum functioning. An important observation was that divalent cations induce significant interaction between the regulatory and the catalytic domain of MtalphaIPMS thus inducing structural cooperativity in the enzyme. This divalent cation induced structural cooperativity might result in modulation of activity of the catalytic domain by regulatory domain.

Conclusion: The studies for the first time demonstrate that different cations bind at different sites in the enzyme leading to their differential effects on the structure and functional activity of the enzyme.

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Electrostatic potential of molecular surface of MtαIPMS dimer. The colors blue and red represents negative and positive potential. Panels A and B represent MtαIPMS dimer molecule in two different orientations. The molecular surface was displayed using GRASP.
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Figure 4: Electrostatic potential of molecular surface of MtαIPMS dimer. The colors blue and red represents negative and positive potential. Panels A and B represent MtαIPMS dimer molecule in two different orientations. The molecular surface was displayed using GRASP.

Mentions: We wanted to understand the underlying mechanism for differential action of cations on both the structure and function of the MtαIPMS. The electrostatic surface potential of the enzyme as obtained from GRASP shows no large concentration of positive or negative charged regions on the enzyme surface (Figure 4). Hence, non-specific binding of cations on protein surface will be low. Furthermore, a differential effect of activation or inhibition and partial unfolding or no effect on the structure of enzyme by similarly charged cations like Mg2+, Cd2+ and Zn2+ was observed. These observations suggest a possibility of interaction of cations with similar charges at different sites in the enzyme. This possibility is further strengthened by the observation that Cd2+ and Zn2+ induce only partial unfolding but almost complete loss of functional activity of the enzyme. So we concentrated our efforts on mapping site of interaction of various cations on MtαIPMS. To obtain an isolated folded catalytic domain of the enzyme, limited proteolysis technique was applied which has been demonstrated by us to be a technique of choice for obtaining isolated domain/s in folded functionally active form from the full-length protein [11-13].


Cation induced differential effect on structural and functional properties of Mycobacterium tuberculosis alpha-isopropylmalate synthase.

Singh K, Bhakuni V - BMC Struct. Biol. (2007)

Electrostatic potential of molecular surface of MtαIPMS dimer. The colors blue and red represents negative and positive potential. Panels A and B represent MtαIPMS dimer molecule in two different orientations. The molecular surface was displayed using GRASP.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919377&req=5

Figure 4: Electrostatic potential of molecular surface of MtαIPMS dimer. The colors blue and red represents negative and positive potential. Panels A and B represent MtαIPMS dimer molecule in two different orientations. The molecular surface was displayed using GRASP.
Mentions: We wanted to understand the underlying mechanism for differential action of cations on both the structure and function of the MtαIPMS. The electrostatic surface potential of the enzyme as obtained from GRASP shows no large concentration of positive or negative charged regions on the enzyme surface (Figure 4). Hence, non-specific binding of cations on protein surface will be low. Furthermore, a differential effect of activation or inhibition and partial unfolding or no effect on the structure of enzyme by similarly charged cations like Mg2+, Cd2+ and Zn2+ was observed. These observations suggest a possibility of interaction of cations with similar charges at different sites in the enzyme. This possibility is further strengthened by the observation that Cd2+ and Zn2+ induce only partial unfolding but almost complete loss of functional activity of the enzyme. So we concentrated our efforts on mapping site of interaction of various cations on MtαIPMS. To obtain an isolated folded catalytic domain of the enzyme, limited proteolysis technique was applied which has been demonstrated by us to be a technique of choice for obtaining isolated domain/s in folded functionally active form from the full-length protein [11-13].

Bottom Line: The activator K+ also interacts with the catalytic TIM barrel domain however, it does not induce any significant effect on the enzyme structure.This divalent cation induced structural cooperativity might result in modulation of activity of the catalytic domain by regulatory domain.The studies for the first time demonstrate that different cations bind at different sites in the enzyme leading to their differential effects on the structure and functional activity of the enzyme.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow, India. ksinghcdri@gmail.com <ksinghcdri@gmail.com>

ABSTRACT

Background: Alpha-isopropylmalate synthase (MtalphaIPMS), an enzyme that catalyzes the first committed step of the leucine biosynthetic pathway of Mycobacterium tuberculosis is a potential drug target for the anti-tuberculosis drugs. Cations induce differential effect of activation and inhibition of MtalphaIPMS. To date no concrete mechanism for such an opposite effect of similarly charged cations on the functional activity of enzyme has been presented.

Results: Effect of cations on the structure and function of the MtalphaIPMS has been studied in detail. The studies for the first time demonstrate that different cations interact specifically at different sites in the enzyme and modulate the enzyme structure differentially. The inhibitors Zn2+ and Cd2+ ions interact directly with the catalytic domain of the enzyme and induce unfolding/denaturation of the domain. The activator K+ also interacts with the catalytic TIM barrel domain however, it does not induce any significant effect on the enzyme structure. Studies with isolated catalytic TIM barrel domain showed that it can carry out the catalytic function on its own but probably requires the non-catalytic C-terminal domain for optimum functioning. An important observation was that divalent cations induce significant interaction between the regulatory and the catalytic domain of MtalphaIPMS thus inducing structural cooperativity in the enzyme. This divalent cation induced structural cooperativity might result in modulation of activity of the catalytic domain by regulatory domain.

Conclusion: The studies for the first time demonstrate that different cations bind at different sites in the enzyme leading to their differential effects on the structure and functional activity of the enzyme.

Show MeSH
Related in: MedlinePlus