Ha. Koteiche et al., UTILITY OF A NOVEL SPIN-LABELED NUCLEOTIDE IN INVESTIGATION OF THE SUBSTRATE AND EFFECTOR SITES OF PHOSPHORIBULOKINASE, Biochemistry, 34(46), 1995, pp. 15068-15074
The activated spin-label 3-(2-bromoacetamido)proxyl modifies the sulfu
r atom of phosphorothioate-containing AMP, ADP, and ATP analogs in a f
acile reaction that produces a new series of spin-labeled nucleotides.
One of these products, adenosine 5'-O-(S-acetamidoproxyl 3-thiotripho
sphate) (ATP gamma SAP), has been evaluated as a structural probe for
Rhodobacter sphaeroides phosphoribulokinase (PRK). When incubated with
affinity-purified enzyme that contains tightly bound substrate ATP, A
TP gamma SAP binds noncooperatively to the allosteric site (n = 1; K-D
= 8 mu M). Probe bound in this site is displaced (K-1/2 = 100 mu M) b
y the allosteric effector, NADH, at concentrations comparable to those
required for enzyme activation (K-a 133 mu M). In the presence of NAD
H, when PRK's substrate site is vacant, ATP gamma SAP binds in a coope
rative mode (Hill coefficient approximate to 2.9; K-D = 20 mu M). In t
he absence of NADH, ATP gamma SAP mimics ATP by exhibiting nonequilibr
ium binding to PRK. The observations with phosphoribulokinase, togethe
r with the straightforward nature of the methodology documented for sy
nthesis and isolation of this class of spin-labeled nucleotides, sugge
st that these analogs have potentially wide application as structural
probes.