UTILITY OF A NOVEL SPIN-LABELED NUCLEOTIDE IN INVESTIGATION OF THE SUBSTRATE AND EFFECTOR SITES OF PHOSPHORIBULOKINASE

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.