Adenylate kinase from Mycobacterium tuberculosis (AKmt), a protein of 181 residues is short variant with low similarity with the eukaryote cytosolic AKs. NMR and molecular dynamics simulation were used to probe the structural and dynamic behaviors of AKmt and AKmtDM(A11S,E122K). Chemical shift analyses of AKmtDM, using the 1H-15N HSQC experiment, show that ATP binding P-loop(7-13) and LID domain and include LID domain surrounding residues(115~135), are slightly perturbed(δw≈0.1~0.3ppm). NMR results reveal that the backbone structure AKmtDM is differ from AKmt at ATP binding active site and it’s structural perturbation may induce significant backbone rearrangement and eventually produce the difference of catalytic activity. The correlation of binding domain perturbation and chemical shift direction analysis for substrate binding to AKmt and AKmtDM exhibit that ATP binding influence the the AMPbd mobility and finally improve AMP binding affinity to the AMPbd. Dissociation constant analysis show that the binding affinity of ATP to ATPbd in AKmtDM(Kd: 73 ± 33μM) is about 7 fold higher than that of AKmt(498 ± 326μM). Results of backbone dynamics and molecular dynamics simulation suggest that mutations of AKmtDM stabilize Arg123 carrying an important role in catalysis through the charge-charge interactions between E118 and K122 and reinforce the hydrogen bonding network in p-loop, thereby enhance the substrate recognition.