The functions of evolved mammalian supramolecular assemblies and extensions of enzymes are not well understood. Human lysyl-tRNA synthetase (hKRS) only upon the removal of the amino-terminal extension (hKRSDelta60) bound to EF1alpha and was stimulated by EF1alphain vitro. HKRS and hKRSDelta60 were also differentially stimulated by aspartyl-tRNA synthetase (AspRS) from the multi-synthetase complex. The non-synthetase protein from the multi-synthetase complex p38 alone did not affect hKRS lysylation but inhibited the AspRS-mediated stimulation of hKRS. These results revealed the functional interactions of hKRS and shed new lights on the functional significance of the structural evolution of multienzyme complexes and appended extensions.