Direct observation of substrate-enzyme complexation by surface forces measurement.
The substrate-enzyme complexation of heptaprenyl diphosphate synthase was directly investigated using colloidal probe atomic force microscopy (AFM) and a quartz crystal microbalance (QCM) in order to obtain new insights into the molecular mechanism of the enzyme reaction. This enzyme is composed of two dissociable subunits that exhibit a catalytic activity only when they are associated together in the presence of a cofactor, Mg2+, and a substrate, farnesyl diphosphate (FPP). The QCM measurement revealed that FPP was preferentially bound to subunit II in the presence of Mg2+, while the AFM measurement showed that the adhesive force between the subunits was observed only in the presence of both Mg2+ and FPP. This is the first direct demonstration of the specific interaction involved in the enzyme reaction. The dependence of the Mg2+ concentration on the specific interaction between subunits I and II well agreed with that on the enzyme activity of heptaprenyl diphosphate synthase. This indicated that the observed adhesive forces were indeed involved in the catalytic reaction of this enzyme. On the basis of these results, we discussed the processes involved in the substrate-enzyme complexation. The first, the substrate FPP bound to subunit II using Mg2+, followed by the formation of the subunit I-FPP-Mg2+-subunit II complex. Our study showed a very useful methodology for examining the elemental processes of biological reactions such as an enzyme reaction.
Suzuki
Takehiro
T
Institute for Multidisciplinary Research for Advanced Materials, Tohoku University, Aoba-ku, Sendai 980-8577, Japan.
Zhang
Yuan-Wei
YW
Koyama
Tanetoshi
T
Sasaki
Darryl Y
DY
Kurihara
Kazue
K
D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D018625:Microscopy, Atomic Force / Q000379:methods; D011106:Polyisoprenyl Phosphates / Q000737:chemistry* / Q000378:metabolism*; D021122:Protein Subunits; D012717:Sesquiterpenes
D011106:Polyisoprenyl Phosphates; D021122:Protein Subunits; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C024844:trans-hexaprenyltranstransferase; D008274:Magnesium