Table 1. Purification of dihydrodipicolinate synthase from Black Mexican Sweet Corn cultures.
The purified preparation after anion exchange chromatography contained three detectable proteins as visualized on a silver-stained native gel. One of the proteins had an estimated molecular weight of 120,000Mr, which is similar to the estimated molecular weight of DHPS from wheat (Kumpaisel et al., Plant Physiol. 85:145-51, 1987). The purified DHPS had a pH optimum of 8.5 in a tris buffer.
Estimates of the Km values were 0.4mM for aspartate semialdehyde (ASA) and 5mM for pyruvate. Increasing L-ASA concentrations showed substrate inhibition which became less apparent as the concentration of pyruvate or L-lysine increased. The alleviating effect by pyruvate on ASA substrate inhibition indicates an ordered enzyme mechanism, either an ordered bi-bi or ping-pong reaction mechanism with pyruvate binding first.
Lysine inhibition was found to be of a mixed type with respect to L-ASA and of a noncompetitive type with respect to pyruvate. Lysine inhibition showed cooperativity with an I(.5) of 25µM and an estimated Hill number of 2.8, indicating at least three, or more likely four, interacting subunits.
In a survey of lysine analogues, lysine ethyl ester, threo-hydroxylysine, aminoethyl cysteine, and arginine inhibited DHPS 89, 83, 18, and 8%, respectively, at 100µM. Other analogues which did not show inhibition at 100µM were diaminopimelic acid, delta-hydroxylysine, epsilon-caproic acid, aspartate, norleucine and D-lysine. From these analogue studies, it appears that the alpha and epsilon amino groups and their stereochemistry are important in inhibitor binding; whereas, the carboxyl group is less important.
Enzyme preparations from the Ltr*-1 and Ltr*-2 mutants
as described by Dotson et al. (MNL, this issue) also were analyzed for
DHPS activity and inhibition by lysine. DHPS from the two mutants did not
exhibit changes in kinetic parameters or lysine inhibition relative to
the A619 wildtype control, indicating that control of the lysine-specific
biosynthesis pathway was not altered in the mutants.
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