Abstract
Dihydroxy acid dehydratase, the third enzyme in the branched-chain amino acid biosynthetic pathway, has been purified to homogeneity (5000-fold) from spinach leaves. The molecular weights of dihydroxy acid dehydratase as determined by sodium dodecyl sulfate and native gel electrophoresis are 63,000 and 110,000, respectively, suggesting the native enzyme is a dimer. 2 moles of iron were found per mol of protein monomer. Chemical analyses of iron and labile sulfide gave an Fe/S2- ratio of 0.95. The EPR spectrum of dithionite-reduced enzyme (gavg = 1.91) is similar to spectra characteristic of Rieske Fe-S proteins and has a spin concentration of 1 spin/1.9 irons. These results strongly suggest that dihydroxy acid dehydratase contains a [2Fe-2S] cluster, a novel finding for enzymes of the hydrolyase class. In contrast to the Rieske Fe-S proteins, the redox potential of the Fe-S cluster is quite low (-470 mV). Upon addition of substrate, the EPR signal of the reduced enzyme changes to one typical of 2Fe ferredoxins (gavg = 1.95), and the visible absorption spectrum of the native enzyme shows substantial changes between 400 and 600 nm. Reduction of the Fe-S cluster decreases the enzyme activity by 6-fold under Vmax conditions. These results suggest the direct involvement of the [2Fe-2S] cluster of dihydroxy acid dehydratase in catalysis. Similar conclusions have been reached for the catalytic involvement of the [4Fe-4S] cluster of the hydrolyase aconitase (Emptage, M. H., Kent, T. A., Kennedy, M. C., Beinert, H., and Münck, E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4674-4678).
Highlights
These results mechanism presumably would hold for theplant enzyme strongly suggest that dihydroxy acid dehydratase con- because plants do not containcoenzyme BIZ.Dihydroxy acid tains a [2Fe-2S] cluster, a novel finding for enzymes dehydratase from bacteria has beenshown to catalyze the of the hydrolyase class
Fe-S cluster decreases the enzyme activity by 6-fold under V, conditions. These results suggest the direct enolintermediate directly into solution [3].Thereaction catalyzed by dihydroxy acid dehydratase from bacteria occurs with retention of configuration at C-3, i.e. the proton adds to the side of the enolfrom which hydroxyl was eliminated [6]
Purification of DihydroxyAcid Dehydratase-Table I shows the course of a typical purification of spinach dihydroxy acid dehydratase
Summary
Dihydroxy acid reduced enzyme (gave= 1.91) issimilartospectra dehydratase from bacteria (Salmonellatyphimurium) has characteristic of Rieske Fe-S proteins and has a spin been shown to actvia an enol intermediate [4], and the same concentration of 1 spin/l.9 irons. Droxy acid dehydratase hasbeen found in bacteria,fungi, and An obvious color associatedwith the purifiedenzyme green plants and hasbeen partially purified from Escherichia prompted an investigation of its metal content. This led to coli, Neurospora crassa, Spinach oleracea, and Phmeolus ra- the surprisingdiscovery that theenzyme contains a [2Fe-2S].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
