The flesh-eating bacterium group A Streptococcus (GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry (MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of S. pneumoniae -enolase. Site-directed mutagenesis of surface-located lysine residues (SENK252+255A, SENK304A, SENK334A, SENK344E, SENK435L and SEN434-435) was used to examine their roles in maintaining structural integrity, enzymatic function and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SENK344E, as determined by circular dichroism spectroscopy and MS. However, ion-mobility MS revealed distinct differences in the stability of several mutant octamers in comparison to wild-type. Enzymatic analysis indicated that SENK344E had lost -enolase activity, which was also reduced in SENK334A and SENΔ434-435. Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SENK252+255A, SENK435L and SENΔ434-435. The lysine residues at positions 252, 255, 434 and 435, therefore play a concerted role in plasminogen acquisition. This study demonstrates the ability of combining in silico structural modeling with ion mobility-MS validation for undertaking functional studies on complex protein structures.