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 brain UMP/CMP kinase (UCK): one, designated as UAU, carries an AMP-binding 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 revealed that UAU is 4-fold more active for AMP, 40-fold less active for UMP, and 4-fold less active for CMP than the parental UCK, although AUA has considerably lowered reactivity for both AMP and UMP. Circular dichroism spectra of the two chimeric enzymes suggest that UAU and AUA have similar folding structures to UCK and AK, respectively. Furthermore, proton NMR measurements of the UCK and UAU proteins indicate that significant differences in proton signals are limited to the aromatic region, where an imidazole C2H signal assigned to His31 shows a downfield shift upon conversion of UCK to UAU, and the signals assigned to Tyr49 and Tyr56 in the UMP/CMP-binding domain disappear in UAU. In contrast, AUA has a Tm value about 11 degreesC lower than AK, whereas UAU and UCK have similar Tm 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.
Okajima
T
T
Faculty of Agriculture, Kinki University, Nara, 631-8505, Japan. okajima@nara.kindai.ac.jp
Fukamizo
T
T
Goto
S
S
Fukui
T
T
Tanizawa
K
K
D000263:Adenylate Kinase / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002645:Chickens; D002942:Circular Dichroism; D004795:Enzyme Stability; D007700:Kinetics; D008969:Molecular Sequence Data; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D011993:Recombinant Fusion Proteins; C019979:cytidylate kinase; D000263:Adenylate Kinase; D009703:Nucleoside-Phosphate Kinase