Pyridoxal phosphate binding sites are similar in human heme-dependent and yeast heme-independent cystathionine beta-synthases - Evidence from P-31 NMR and pulsed EPR spectroscopy that heme and PLP cofactors are not proximal in the human enzyme
O. Kabil et al., Pyridoxal phosphate binding sites are similar in human heme-dependent and yeast heme-independent cystathionine beta-synthases - Evidence from P-31 NMR and pulsed EPR spectroscopy that heme and PLP cofactors are not proximal in the human enzyme, J BIOL CHEM, 276(22), 2001, pp. 19350-19355
Two classes of cystathionine P-synthases have been identified in eukaryotes
, the heme-independent enzyme found in yeast and the heme-dependent form fo
und in mammals. Both classes of enzymes catalyze a pyridoxal phosphate (PLP
)-dependent condensation of serine and homocysteine to produce cystathionin
e. The role of the heme in the human enzyme and its location relative to th
e PLP in the active site are unknown. P-31 NMR spectroscopy revealed that s
pin-lattice relaxation rates of the phosphorus nucleus in PLP are similar i
n both the paramagnetic ferric (T-1 = 6.34 +/- 0.01 s) and the diamagnetic
ferrous (T-1 = 5.04 +/- 0.06 s) enzyme, suggesting that the two cofactors a
re not proximal to each other. This is also supported by pulsed EPR studies
that do not provide any evidence for strong or weak coupling between the p
hosphorus nucleus and the ferric iron. However, the P-31 Signal in the redu
ced enzyme moved from 5.4 to 2.2 ppm, and the line width decreased from 73
to 16 Hz, providing the first structural evidence for transmission to the a
ctive site of an oxidation state change in the heme pocket. These results a
re consistent with a regulatory role for the heme as suggested by previous
biochemical studies from our laboratory. The P-31 chemical shifts of the re
sting forms of the yeast and human enzymes are similar, suggesting that des
pite the difference in their heme content, the microenvironment of the PLP
is similar in the two enzymes. The addition of the substrate, serine, resul
ted in an upfield shift of the phosphorus resonance in both enzymes, signal
ing formation of reaction intermediates. The resting enzyme spectra were re
covered following addition of excess homocysteine, indicating that both enz
ymes retained catalytic activity during the course of the NMR experiment.