Figure 1.
A) Reactions in B6 metabolism. Reaction 1, enzymes involved in the de novo biosynthesis of pyridoxine 5′-phosphate (PNP); Reaction 2, PNP oxidase; Reaction 3, reaction of apo-B6 enzymes with PLP to form active holo-B6 enzymes; Reaction 4, degradation of holo-B6 enzymes to amino acids and PLP; Reaction 5, PLP phosphatase; Reaction 6, PL kinase. B) Structures of B6 vitamers.
Figure 2.
Kinetics of ePL kinase inhibition.
The formation of PLP by ePL kinase was followed at 388 nm in 400 µl of reaction solution containing 1 mM MgATP, 0.2 mM MgCl2, and 1 mM PL at 37°C. At the first arrow, ePL kinase was added to 0.9 µM and PLP formation followed for about 120 seconds. A second aliquot of ePL kinase was added at the second arrow.
Figure 3.
Formation of an ePL kinase•PLP complex.
ePL kinase (300 µM) was incubated with MgATP (1 mM), PL (400 µM) and MgCl2 (0.2 mM) for 1 hour at 37°C in a volume of 2 ml. Then it was added to a 0.6 mm×45 cm column of Sephadex G-50 equilibrated with 1 mM MgATP in reaction buffer and eluted with equilibration solution. Aliquots of 400 µl were collected and absorbance at 278 nm (ePL kinase) and 388 nm (PLP) was recorded. Inset: spectrum of ePL kinase with bound PLP showing that the bound PLP exhibits absorbance peaks at 336 nm and 420 nm.
Figure 4.
Rate of formation of ePL kinase•PLP complex.
To a series of 100 µl solutions in Eppendorf vials containing 0.4 mM MgATP, 0.20 mM MgCl2 and either 0.150 mM PL or 0.150 mM PLP at 37°C was added 9 nmoles of ePL kinase (90 µM). After 2, 5, 15 and 25 min contents of vials were withdrawn and placed on small Sephadex G-50 columns at 4°C equilibrated with 1 mM MgATP and 0.2 mM MgCl2 (see Experimental Procedures) to separate bound and free PLP. The eluate of each column was 400 µl. Spectra were recorded and the absorbance at 420 nm determined. Open circles, reactions initiated with PL, closed circles reactions initiated with PLP. The lines through the experimental points are those obtained from nonlinear least squares fittings of data to an exponential equation which gave rate constants of 0.4 min−1 and 0.1 min−1 and amplitudes of 100% and 83% for the experiments initiated with PL and PLP, respectively.
Table 1.
Rate of formation of ePL kinase•PLP complex in the presence of different substrates and productsa.
Table 2.
Measurement of nucleotides in the ePL kinase•PLP complexesa.
Figure 5.
Rate of dissociation of PLP from the ePL kinase•PLP complex.
The rate of dissociation of PLP from the ePL kinase•PLP complex was followed by observing the change in optical activity of the bound PLP at 37°C. Panel A: Spectra of the complex (60 µM) at time zero (curve a) and after 120 min in the presence of 10 µM PLP phosphatase (curve b). Panel B: measured decrease in optical activity after addition of PLP phosphatase at 415 nm with time and as an exponential process with rate constant of 0.012 min−1.
Figure 6.
Mechanism of reaction between PLP and the active site K229.
A) A scheme showing the structures of the carbinolamine intermediate and the enolimine form of the protonated PLP aldimine. B) Active site structure of the binary complex of ePL kinase and PL showing the position of K229.
Table 3.
Kinetic parameters for wild type and K229Q mutant ePL kinase.
Figure 7.
Comparison of PLP formation with wild type and K229Q ePL kinases.
The kinetics of PLP formation catalyzed by ePL kinase was followed at 388 nm upon addition (shown by the arrow) of 0.3 µM of wild type enzyme (continuous line) or 0.3 µM of K229Q enzyme (dotted line). Each reaction contained 1 mM MgATP, 0.2 mM MgCl2 and 1 mM PL, at 37°C.
Figure 8.
Rate of transfer of PLP from PL kinase•PLP to apo E. coli serine hydroxymethyltransferase.
Fraction of apo-eSHMT (20 µM) being converted to holo-eSHMT with PLP (20 µM) (•–•). Fraction of apo-eSHMT (20 µM) being converted to holo-eSHMT with an equivalent amount of PL kinase•PLP (20 µM) (▴–▴). Repeat of the conversion of apo-eSHMT with free PLP (○–○) or PL kinase•PLP (Δ–Δ) to holo-eSHMT in the presence of 3 µM PLP phosphatase.