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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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Related in: MedlinePlus

Limited proteolysis of MtαIPMS with α-chymotrypsin at pH 7.5 and 25°C. SDS-PAGE profile of the protein fragment(s) obtained on limited proteolysis of recombinant MtαIPMS with α-chymotrypsin. Lanes 1–4 represent molecular weight markers, undigested MtαIPMS, protein digested with α-chymotrypsin and purified Band I, respectively.
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Figure 5: Limited proteolysis of MtαIPMS with α-chymotrypsin at pH 7.5 and 25°C. SDS-PAGE profile of the protein fragment(s) obtained on limited proteolysis of recombinant MtαIPMS with α-chymotrypsin. Lanes 1–4 represent molecular weight markers, undigested MtαIPMS, protein digested with α-chymotrypsin and purified Band I, respectively.

Mentions: Figure 5 summarizes the SDS-PAGE profile of the protein fragments obtained on limited proteolysis of recombinant MtαIPMS with α-chymotrypsin. A major band (Band I) along with three minor bands of lower molecular weight proteins/fragments were observed. The Band I purified by SEC1 showed a molecular mass of 46.6 kDa (as determined by ESI-MS). There are about 47 cleavage sites for α-chymotrypsin spread all over the primary sequence of MtαIPMS however, on limited proteolysis, an intact domain of about 46 kDa was obtained. This suggests that the 46 kDa domain obtained on limited proteolysis of full length protein is in a folded conformation as several of the proteolytic sites present in it are buried in protein interior and not accessible to protease for cleavage under experimental conditions.


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

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

Limited proteolysis of MtαIPMS with α-chymotrypsin at pH 7.5 and 25°C. SDS-PAGE profile of the protein fragment(s) obtained on limited proteolysis of recombinant MtαIPMS with α-chymotrypsin. Lanes 1–4 represent molecular weight markers, undigested MtαIPMS, protein digested with α-chymotrypsin and purified Band I, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Limited proteolysis of MtαIPMS with α-chymotrypsin at pH 7.5 and 25°C. SDS-PAGE profile of the protein fragment(s) obtained on limited proteolysis of recombinant MtαIPMS with α-chymotrypsin. Lanes 1–4 represent molecular weight markers, undigested MtαIPMS, protein digested with α-chymotrypsin and purified Band I, respectively.
Mentions: Figure 5 summarizes the SDS-PAGE profile of the protein fragments obtained on limited proteolysis of recombinant MtαIPMS with α-chymotrypsin. A major band (Band I) along with three minor bands of lower molecular weight proteins/fragments were observed. The Band I purified by SEC1 showed a molecular mass of 46.6 kDa (as determined by ESI-MS). There are about 47 cleavage sites for α-chymotrypsin spread all over the primary sequence of MtαIPMS however, on limited proteolysis, an intact domain of about 46 kDa was obtained. This suggests that the 46 kDa domain obtained on limited proteolysis of full length protein is in a folded conformation as several of the proteolytic sites present in it are buried in protein interior and not accessible to protease for cleavage under experimental conditions.

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