Physicochemical consequences of the perdeuteriation of glutathione S-transferase from S. japonicum.
Glutathione S:-transferase (GST) from Schistosoma japonicum has been prepared in both normal protiated (pGST) and fully deuteriated (dGST) form by recombinant DNA technology. Electrospray mass spectrometry showed that the level of deuteriation in dGST was 96% and was homogeneous across the sample. This result is attributed to the use of a deuterium-tolerant host Escherichia coli strain in the preparation of the protein. 10 heteroatom-bound deuteriums (in addition to the carbon-bound deuteriums) were resistant to exchange when dGST was incubated in protiated buffer. The physicochemical and biological properties of the two proteins were compared. dGST was relatively less stable to heat denaturation and to proteolytic cleavage than was pGST. The midpoint transition temperature for pGST was 54.9 degrees C, whereas that for dGST was 51.0 degrees C. Static light-scattering measurements revealed that the association behavior of dGST is also different from that of pGST. The perdeuteriated enzyme shows a tendency to associate into dimers of the fundamental dimer. This is in contrast with results that have been obtained for other perdeuteriated proteins in which perdeuteriation has been shown to promote dissociation of aggregates. dGST showed a similar K(m) to pGST; similar results had been obtained previously with bacterial alkaline phosphatase. However, whereas the alkaline phosphatase showed a reduced rate of catalysis on deuteriation, dGST exhibited a slightly higher rate of catalysis than pGST. It is clear that the bulk substitution of deuterium for protium has significant effects on the properties of proteins. Until many more examples have been studied, it will be difficult to predict these effects for any given protein. Nevertheless, deuteriation represents an intriguing method of preparing functional analogs of recombinant proteins.
Brockwell
D
D
School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, M13 9PL, UK.
Yu
L
L
Cooper
S
S
McCleland
S
S
Cooper
A
A
Attwood
D
D
Gaskell
S J
SJ
Barber
J
J
D000818:Animals; D002152:Calorimetry, Differential Scanning; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D003903:Deuterium / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D005982:Glutathione Transferase / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D006358:Hot Temperature; D006868:Hydrolysis; D007554:Isotopes / Q000737:chemistry / Q000378:metabolism; D012549:Schistosoma japonicum / Q000201:enzymology*; D021241:Spectrometry, Mass, Electrospray Ionization
D007554:Isotopes; D003903:Deuterium; D005982:Glutathione Transferase