Sulfhydryl groups in relation to the structure and catalytic activity of 2-OXO-4-hydroxyglutarate aldolase from bovine liver
Lane, Roger S.; Hansen, Barbara A.; Dekker, Eugene E.
1977-03-15
Citation
Lane, Roger S., Hansen, Barbara A., Dekker, Eugene E. (1977/03/15)."Sulfhydryl groups in relation to the structure and catalytic activity of 2-OXO-4-hydroxyglutarate aldolase from bovine liver." Biochimica et Biophysica Acta (BBA) - Enzymology 481(1): 212-221. <http://hdl.handle.net/2027.42/22958>
Abstract
Bovine liver 2-oxo-4-hydroxyglutarate aldolase (suggested name: 2-oxo-4hydroxyglutarate glyoxylate-lyase catalyzing the reaction: 2-oxo-4-hydroxyglutarate rlhar2 pyruvate + glyoxylate) contains eight to ten sulfhydryl groups as determined by titration of the enzyme with either 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) or p-mercuribenzoate in the presence of 1% sodium dodecyl sulfate. In the absence of a denaturant, all of the cysteinyl residues react with p-mercuribenzoate whereas only four are accessible to titration with Nbs2. No differences in -SH group reactivity can be detected during titration of the aldolase with p-mercuribenzoate. In contrast, two classes of sulfhydryls can be differentiated in the disulfide exchange reaction with Nbs2 in the absence of a denaturant; one -SH group (Class I) reacts rapidly whereas three additional thiols (Class II) titrate at approx. 0.1 the rate of the Class I -SH residue. Both pyruvate and glyoxylate protect one of the three -SH residues in Class II from reaction with Nbs2. Either substrate also prevents titration of one to two thiol groups by p-mercuribenzoate and decreases the rate of reaction of aldolase -SH groups with Nbs2 in 8 M urea. These ligand-induced changes in -SH reactivity provide a sensitive indication that the enzyme exists in an altered conformational state in the presence of either of its cosubstrates.Titration of the enzyme with either Nbs2 or p-mercuribenzoate results in a progressive loss of aldolase activity which is not proportional to the number of -SH groups modified. The enzyme retains 50% of the activity of the native enzyme when Class I and Class II thiols (i.e. four -SH groups total) are modified with Nbs2; 15% residual activity is still observed following titration of all of the cysteinyl residues with p-mercuribenzoate. Pyruvate and glyoxylate provide partial protection against inactivation. It is concluded that inactivation of 2-oxo-4-hydroxyglutarate aldolase by Nbs2 or p-mercuribenzoate is a consequence of alterations in protein structure which accompany modification of -SH groups. The data argue against the direct participation of an active-site thiol group in the catalytic mechanism of 2-oxo-4-hydroxyglutarate aldolase, be that aldol cleavage and condensation or [beta]-decarboxylation.Publisher
Elsevier
PMID
557345
Types
Article
URI
http://www.sciencedirect.com/science/article/B73GH-47SV4DJ-1P/2/11e259f6cb38d8cb6d199eb2e12273bdhttp://www.ncbi.nlm.nih.gov/sites/entrez?cmd=retrieve&db=pubmed&list_uids=557345&dopt=citation
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