Aa. Rezende et al., PHOSPHODIESTERASE ACTIVITY IS A NOVEL PROPERTY OF ALKALINE-PHOSPHATASE FROM OSSEOUS PLATE, Biochemical journal, 301, 1994, pp. 517-522
Phosphodiesterase activity is a novel property of the still-enigmatic
alkaline phosphatase from osseous plate. Bis-(p-nitrophenyl) phosphate
was hydrolysed at both pH 7.5 and 9.4 with an apparent dissociation c
onstant (K (0.5)) of 1.9 mM and 3.9 mM respectively. The hydrolysis of
p-nitrophenyl-5'-thymidine phosphate followed hyberbolic kinetics wit
h a K-0.5 of 500 mu M. For p-nitrophenyl phenylphosphonate, site-site
interactions [Hill coefficient (h) = 1.3] were observed in the range b
etween 0.2 and 100 mu M, and K-0.5 was 32.8 mM. The hydrolysis of cycl
ic AMP by the enzyme followed more complex kinetics, showing site-site
interactions (h = 1.7) and K-0.5 = 300 mu M for high-affinity sites.
The low-affinity sites, representing 85% of total activity, also showe
d site-site interactions (h = 3.8) and a K-0.5 of about 22 mM. ATP and
cyclic AMP were competitive inhibitors of bis-(p-nitrophenyl) phospha
tase activity of the enzyme and K-1 values (25 mM and 0.6 mM for cycli
c AMP and ATP respectively) very close to those of the K-0.5 (22 mM an
d 0.7 mM for cyclic AMP and ATP respectively), determined by direct as
say, indicated that a single catalytic site was responsible for the hy
drolysis of both substrates. Non-denaturing PAGE of detergent-solubili
zed enzyme showed coincident bands on the gel for phosphomonohydrolase
and phosphodiesterase activities. Additional evidence for a single ca
talytic site was the similar pK(a) values (8.5 and 9.7) found for the
two ionizing groups participating in the hydrolysis of bis-(p-nitrophe
nyl) phosphate and p-nitrophenyl phosphate. The alkaline apparent pH o
ptima, the requirement for bivalent metal ions and the inhibition by m
ethylxanthines, amrinone and amiloride demonstrated that rat osseous-p
late alkaline phosphatase was a type I phosphodiesterase. Considering
that there is still confusion as to which is the physiological substra
te for the enzyme, the present results describing a novel property for
this enzyme could be of relevance in understanding the mineralization
process.