Carbohydrate transfer reactions are significant for organisms and are involved in a variety of functions in cells. This complicated system is controlled by enzymes including glycosyltransferases (GTs). GTs catalyze sugar transfer reactions. There are mainly two structural folds adopted by GTs, GT-A and GT-B. Structures of the GT-B enzymes are well conserved, especially in the C-terminal domain. GT-Bs are believed to undergo a conformational change upon binding substrates. This indicates that protein dynamics of GT-B members is a key for the study of the whole family. To better understand the commonly shared conformational change by GT-B enzymes, backbone amide hydrogen-deuterium exchange monitored by mass spectrometry (HDX-MS) is utilized to characterize protein motion in solution. We report results of HDX-MS experiments to determine conformational changes of two representatives of GT-B enzymes, MshA from Corynebacterium glutamicum (CgMshA) and Heptosyltransferase I (HepI). HDX-MS analysis of CgMshA suggests a third conformation of CgMshA in solution with uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc) bound. It indicates that the UDP-GlcNAc complex might be in a loose form compared with UDP complex, and the UDP release step might be rate determining for the reaction. Moreover, structural and dynamic studies are combined with bioinformatic results for CgMshA to predict the sequence/structure/dynamic relationships for the GT4 family. In addition, protein dynamics of HepI in the apo form is studied and compared to CgMshA. HDX-MS analysis of HepI suggests that the regions in the outer layer of HepI exhibit flexibility, which predicts a slight domain rotation in HepI. The flexible regions of HepI might participate in the potential conformational change. Other contributions of an additional domain are investigated in this dissertation in terms of protein activity and mechanism. By studying isopropylmalate synthase and citramalate synthase from Methanococcus jannaschii (MjIPMS and MjCMS, respectively), the role of the LeuA dimer regulatory domain in substrate selectivity is determined. Overall, we report a more complex role for the LeuA dimer regulatory domain in substrate selectivity through catalytic modulations rather than selectivity through differential binding as a result of extensive co-evolution between the catalytic and regulatory domains.