Remarkably, it was discovered that 1 undergoes rapid addition of water at the phosphorus center to form 3, with the reaction proceeding quantitatively in the presence of a single equivalent of substrate (Scheme 3).
Evidence that both ferric and ferrous oxidation states of iron are involved in the lipoxygenase catalytic cycle came initially from observation of a high spin iron-like EPR signal after the enzyme turned over a single equivalent of substrate, or was exposed to an equivalent of its own product, a lipid allylic hydroperoxide.
(1) In contrast to 2, when the reaction is monitored by P NMR spectroscopy, the observed product is 4-nitrophenyl phosphate monoester, with no evidence of a transesterification intermediate, and the complex completely hydrolyzes 5 equivalents of substrate (no substrate observable after 50 days), thus demonstrating catalytic turnover.
This suggests that extra HdrABC may circumvent the production of a transmembrane ion gradient, leading to decreased ATP generation, and consequently may result in decreased cell growth from an equivalent amount of substrate.
The analysis results revealed that the BGL activity peaked by 120 h culture, releasing 0.39 ± 0.02 mg glucose in the above reaction mixture, which is equivalent to 10% of substrate cellobiose being hydrolyzed into glucose within the first hour of hydrolysis.
The clearly understandable differences between the first "half" of the catalytic cycle, visualized in Figure 19, I, and the "second half", Figure 19, II, arise because the former involves accumulation of reducing equivalents while the latter involves delivery of reducing equivalents to substrate.
Two cysteines in this motif were the source of reducing equivalents for substrate reduction.
One molar equivalent of acyl chloride substrate was reacted with 15 molar equiv of H2O in the presence of dried pyridine for 10 min in a nitrogen-sparged flask.
Once cooled the substrate was charged with Cellic CTec3 enzyme at a concentration of 50 55 mg per g of substrate, equivalent to 66.56.5 FPU g-1 dry substrate.
But this need not be equivalent to transmembrane movement of substrate, which necessarily involves a steady-state cycling of the protein through multiple conformations (inward-facing, empty – inward-facing, substrate bound, outward-facing, substrate bound, inward occluded, etc).
It is the functional equivalent of human NAT1; the substrate specificity profile of mouse Nat2 is like that of human NAT1 (Kawamura et al., 2008): the C-terminal sequence is the same as human NAT1 and mouse NAT2 is also recognized by human NAT1-specific antibodies raised against the C-terminus.
