Recent studies have revealed that mutations of residues distal to the enzymatic active site of DHFR can change enzymatic activity without interacting directly with the substrate. Although the mutations were of noncatalytic residues, they affected catalysis by altering conformational and dynamical states of the active site. Toward this end, we have employed site-directed chemical cross-linking (SDXL) to control loop dynamics and conformations of dihydrofolate reductase (DHFR) from Escherichia coli. Previous studies have indicated the Met20, the F-G and the G-H loops play central roles in the catalytic activity and mechanism of DHFR. In this work, we show that inserting chemical cross-links at specific locations between the Met20 and F-G loops of DHFR modulates DHFR activity in a cross-link length dependent fashion. The extent to which crosslinking modulates DHFR activity by altering local conformational dynamics of secondary structure motifs was examined using hydrogen/deuterium exchange mass spectrometry (HDX-MS). Three regions of DHFR [residues 9-24 (Met20 loop), 116-132 (F-G loop), and 25-36 (αB, active site)] were found to have altered exchange properties upon cross-linking. These results are consistent with observed changes in enzymatic activity and substrate binding affinity (KD) upon cross-linking. The data illustrates SDXL as a means for targeting and perturbing specific protein dynamics, even at locations distal to the active site, to modulate enzymatic activity.