Studies of substrate-induced conformational changes in human cytomegalovirus protease using optical biosensor technology.
The interaction between human cytomegalovirus (HCMV) protease and a peptide substrate was studied using a surface plasmon resonance (SPR)-based biosensor. Immobilization of the enzyme to the sensor chip surface by amine coupling resulted in an active enzyme with a higher catalytic efficiency than the enzyme in solution, primarily due to a lower K(m) value. The interaction between immobilized protease and substrate was characterized by a biphasic SPR signal. Rate constants for the formation of the initial enzyme-substrate complex could be determined from the sensorgrams. Simulated binding curves based on the determined k(cat) and the rate constants indicated that the complex binding signal did not originate from the accumulation of intermediates in the catalytic reaction. By chemical crosslinking of the immobilized HCMV protease, which was shown to limit the enzyme's structural flexibility, it was revealed that the obtained sensorgrams were composed of a signal caused by substrate binding and considerable structural alterations in the immobilized enzyme. Furthermore, HCMV protease was inactivated by chemical crosslinking, indicating that structural flexibility is essential for this enzyme. Parallel experiments with immobilized alpha-chymotrypsin revealed that it does not undergo similar conformational changes on peptide binding and that crosslinking did not inactivate the enzyme. The simultaneous detection of binding and conformational changes using optical biosensor technology is expected to be of importance for further characterization of the enzymatic properties of HCMV protease and for identification of inhibitors of this enzyme. It can also be of use for studies of other flexible proteins.
Department of Biochemistry, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden.
Matthis
M
Geitmann
U Helena
UH
Danielson
D002384:Catalysis; D002918:Chymotrypsin / Q000378:metabolism; D003587:Cytomegalovirus / Q000201:enzymology*; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D020349:Surface Plasmon Resonance / Q000379:methods*
D004800:Enzymes, Immobilized; D012697:Serine Endopeptidases; C071910:assemblin; D002918:Chymotrypsin; C117608:alpha-chymotrypsin