Isocitrate lyase (ICL), a potential anti-tubercular drug target, catalyzes the first step of the glyoxylate shunt. In the present investigation, we studied the conformational flexibility of MtbICL to better understand its stability and catalytic activity. Our biochemical results showed that a point mutation at Phe345, which is topologically distant (>10 Å) to the active site signature sequence (189KKCGH193), completely abolishes the activity of the enzyme. In depth computational analyses were carried out for understanding the structural alterations using molecular dynamics, time-dependent secondary structure and principal component analysis. The results showed that the mutated residue increased the structural flexibility and induced conformational changes near the active site (residues 170-210) and in the C-terminal lid region (residues 411-428). Both these regions are involved in the catalytic activity of MtbICL. Upon mutation, the residual mobility of the enzyme increased, resulting in a decrease in the stability, which was confirmed by the lower free energy of stabilization in the mutant enzyme suggesting the destabilization in the structure. Our results have both biological importance and chemical novelty. It reveals internal dynamics of the enzyme structure and also suggests that regions other than the active site should be exploited for targeting MtbICL inhibition and development of novel anti-tuberculosis compounds.