Construction, separation and properties of hybrid hexamers of glutamate dehydrogenase in which five of the six subunits are contributed by the catalytically inert D165S.
In vitro subunit hybridization was used to explore the basis of putative allosteric behaviour in clostridial glutamate dehydrogenase. C320S and D165S mutant enzymes were chosen to construct the hybrid proteins. The C320S mutant protein is fully active and shows normal allosteric properties but lacks the reactive cysteine. D165S is capable of binding both glutamate and NAD(+) but is catalytically inactive. The mutant proteins were denatured separately in 4 M urea, mixed in a 5 : 1 (D165S/C320S) ratio and diluted into a refolding mixture composed of 2 mM NAD(+), 1 M fluoride and artificial chaperones (4 mM polyoxyethylene 10 lauryl ether and 1.6 mM beta-cyclodextrin). Under these conditions approximately 50% refolding was achieved for both mutant proteins separately. The renatured mixture was concentrated and separated from denatured proteins and the components of the refolding mixture by ultrafiltration and ion-exchange chromatography. Ellman's reagent, 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), which binds close to the NAD(+) binding site, thus abolishing coenzyme binding in the wild-type enzyme, also reacts with D165S but has no effect on C320S. Modification by DTNB was coupled with dye-ligand affinity chromatography on a Procion Red HE-3B column in order to separate the hybrid mixture into fractions of defined composition. An optimized procedure based on salt gradient elution was developed. DTNB-modified 5 : 1 hybrids, with only one subunit capable of binding coenzyme, showed classical Michaelis-Menten kinetics when the NAD(+) concentration was varied, whereas removal of the thionitrobenzoate moieties that blocked the other five coenzyme binding sites in the hexamer reinstated nonlinear behaviour, suggesting that 'nonlinear' behaviour of the native enzyme and the hybrid with six coenzyme binding sites depends on binding to multiple sites. When assayed at high pH with increasing glutamate concentration, the sample with only one active subunit showed reduced sigmoidicity in the dependence of reaction rate on glutamate concentration (h = 3.0) compared with native C320S with six active subunits (h = 5.2) suggesting that the interaction between the subunits was reduced but not abolished completely. Catalytically silent subunits can thus still contribute to cooperativity.
Department of Biochemistry and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland.
B M
BM
Hayden
P C
PC
Engel
D000494:Allosteric Regulation / Q000187:drug effects; D000495:Allosteric Site; D019943:Amino Acid Substitution / Q000235:genetics*; D002384:Catalysis / Q000187:drug effects; D002846:Chromatography, Affinity; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D004396:Coloring Agents / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D009154:Mutation / Q000235:genetics; D009243:NAD / Q000378:metabolism; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D020673:Protein Renaturation / Q000187:drug effects; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014227:Triazines / Q000378:metabolism; D014462:Ultrafiltration; D014508:Urea / Q000494:pharmacology
D004396:Coloring Agents; D008024:Ligands; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D014227:Triazines; D009243:NAD; C003213:Procion Brilliant Red M-2BS; D018698:Glutamic Acid; D014508:Urea; D004228:Dithionitrobenzoic Acid; D005969:Glutamate Dehydrogenase; D003545:Cysteine