EXCHANGE OF NUCLEOSIDE MONOPHOSPHATE-BINDING DOMAINS IN ADENYLATE KINASE AND UMP CMP KINASE/

Two types of active chimeric enzymes have been constructed by genetic engineering of chicken cytosolic adenylate kinase (AK) and porcine bra in UMP/CMP kinase (UCK): one, designated as UAU, carries an AMP-bindin g domain of AK in the remaining body of UCK; and the other, designated as AUA, carries a UMP/CMP-binding domain of UCK in the remaining body of AK, Steady-state kinetic analysis of these chimeric enzymes reveal ed that UAU is 4-fold more active for AMP, LO-fold less active for UMP , and 4-fold less active for CMP than the parental UCK, although AUA h as considerably lowered reactivity for both AMP and UMP. Circular dich roism spectra of the two chimeric enzymes suggest that UAU and AUA hav e similar folding structures to TICK and AK, respectively, Furthermore , proton NMR measurements of the UCK and UAU proteins indicate that si gnificant differences in proton signals are limited to the aromatic re gion, where an imidazole C2H signal assigned to His31 shows a downfiel d shift upon conversion of UCK to UAU, and the signals assigned to Tyr 49 and Tyr56 in the UMP/CMP-binding domain disappear in UAU, In contra st, AUA has a T-m value about 11 degrees C lower than AK, whereas UAU and UCK have similar T-m values. These results together show that the substrate specificity of nucleoside monophosphate (NMP) kinases can be engineered by the domain exchange, even though the base moiety of NMP appears to be recognized cooperatively by both the NMP-binding domain and the MgATP-binding core domain.