The ability of structural analogues of ascorbate to serve as substitutes for this reducing agent in the prolyl 4-hydroxylase reaction was studied. In experiments using the purified enzyme, variations of the compounds' side chain were compatible with co-substrate activity. The presence of very large hydrophobic substituents or a positively charged group caused an increase in the observed Km values. A negative charge and smaller modifications did not change the affinity to the enzyme when compared with L-ascorbate. 6-Bromo-6-deoxy-L-ascorbate had a lower Km than the physiological reductant. Substitution at the -OH group in ring position 3 prevented binding to the enzyme. The same pattern of activity was observed when the full and uncoupled prolyl 4-hydroxylase reactions were studied. The Vmax. values with all compounds were similar. The reaction of microsomal prolyl 4-hydroxylase was supported by D-isoascorbate, O6-tosyl-L-ascorbate and 5-deoxy-L-ascorbate, giving the same dose-response behaviour as L-ascorbate itself. Again, 6-bromo-6-deoxy-L-ascorbate gave a lower Km and a similar Vmax. value. L-Ascorbic acid 6-carboxylate produced substrate inhibition at concentrations above 0.3 mM. The Km and Vmax. values calculated from concentrations up to 0.2 mM were similar to those of L-ascorbate. The enzyme activity observed with 6-amino-6-deoxy-L-ascorbate was very low in the microsomal hydroxylation system. The calculated Vmax. value was lower than that of L-ascorbate, suggesting a restriction of the access of this compound to the enzyme.
ADPGlucose Pyrophosphorylase (ADPG PPase) is an allosteric enzyme that catalyzes the rate‐limiting step in glucan synthesis, an attractive target for engineering to increase the production of renewable carbon. The thermophillic bacterial enzyme from Thermus thermophilus was found to be activated by G6P, F6P, and FBP and displayed optimal activity at 75°C. Based on alignment and molecular modeling studies, R26 and R38 were proposed to be invovled in allosteric regulation of this enzyme. Altered proteins (R26A and R38A) were generated, expressed, and purified. R26A and R38A enzymes displayed a significant decrease in Vmax and a decrease in the apparent affinity for substrates compared to the wild‐type enzyme. In contrast to the wild‐type, the presence of activators caused an increased apparent affinity for the substrate ATP for both enzymes. The R26A mutation resulted in an ~8‐fold decrease in apparent affinity for G6P at 37°C, and a ~30‐fold decrease at 75°C. The R38A enzyme showed ~6‐fold and ~8‐fold decreases in apparent affinity for G6P and F6P at 37°C, respectively. Preliminary data shows decreased apparent affinity for substrates at 75°C for both enzymes. The diminution of the Vmax is consistent with a role for these arginines in stabilizing an active conformation of the enzyme. In addition, R26 appears to play a role in the binding of G6P while R38 appears to facilitate binding of both G6P and F6P in the allosteric subsite(s). Complete kinetic characterization of the altered enzymes and double mutant R26A, R38A at 75°C is underway. Supported by NSF Award 0448676.
ADP‐glucose pyrophosphorylase (ADPG PPase) catalyzes the rate limiting step in glucan synthesis pathways of bacteria and plants and is an attractive target for protein engineering to increase the production of renewable carbon. Agrobacterium tumefaciens (Ag.t.) ADPG PPase is activated by F6P and pyruvate and inhibited by Pi; these effectors are believed to bind at allosteric site(s) located between the catalytic N‐terminal and C‐terminal domains. We probed the role of highly conserved P288 in the loop region between the two domains that has shown to be vital in regulation of the E. coli ADPG PPase. The P288A and P288D mutants were successfully expressed and purified while P288G was found to be unstable. P288A displayed a decrease in activity and fold activation by activators versus wild‐type while the P288D enzyme showed ~5‐fold higher activity than wild‐type and insensitivity to activation and inhibition by effectors. The mutations may alter the loop conformation that positions the domains, resulting in enzyme activation, particularly when negative charge is at this site. Molecular modeling indicates the loop region may interact with another subunit in the tetrameric structure of the enzyme. Efforts are underway to test this hypothesis by additional mutagenesis and structural studies. Supported by NSF Award 0448676.
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