Dehydroquinate synthase: the role of divalent metal cations and of nicotinamide adenine dinucleotide in catalysis.
The cofactor requirements of dehydroquinate synthase from Escherichia coli have been characterized. The homogeneous enzyme, purified from the overproducing strain RB791 (pJB14), is a monomeric metalloenzyme of Mr = 39,000 that contains 1 mol of tightly bound Co(II) according to atomic absorption analysis. The holoenzyme rapidly loses activity upon incubation with EDTA, giving rise to a stable but catalytically inactive apoenzyme. Activity is fully restored by reconstitution with Co(II) and partially restored with other divalent cations. Reconstitution of the apoenzyme with Zn(II) (which is probably the functioning metal in vivo) restores activity to 53% of the level observed with the Co(II)-holoenzyme. The presence of the substrate 3-deoxy-D-arabino-heptulosonate 7-phosphate (1) blocks the inactivation by EDTA. Dehydroquinate synthase also binds 1 mol of NAD+, the presence of which is essential for catalytic activity. The rate constant for the dissociation of NAD+ from the Co(II)-holoenzyme was found to be 0.024 min-1. Under turnover conditions with saturating levels of substrate, the dissociation rate of NAD+ increases by a factor of 40, to 1 min-1. Under these conditions (pH 7.5, 20 degrees C), the Km for NAD+ was determined to be 80 nM.
Bender
S L
SL
Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138.
Mehdi
S
S
Knowles
J R
JR
D001051:Apoenzymes / Q000378:metabolism*; D001059:Apoproteins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002384:Catalysis; D002413:Cations, Divalent / Q000494:pharmacology*; D003035:Cobalt / Q000494:pharmacology; D004492:Edetic Acid; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D009243:NAD / Q000502:physiology*; D019761:Phosphorus-Oxygen Lyases; D013054:Spectrophotometry, Atomic; D013403:Sugar Phosphates / Q000378:metabolism
D001051:Apoenzymes; D001059:Apoproteins; D001426:Bacterial Proteins; D002413:Cations, Divalent; D013403:Sugar Phosphates; D009243:NAD; C013469:3-deoxy-D-arabino-heptulosonate-7-phosphate; D003035:Cobalt; D004492:Edetic Acid; D008190:Lyases; C020001:3-dehydroquinate synthetase; D019761:Phosphorus-Oxygen Lyases