Utility of a Novel Spin-Labeled Nucleotide in Investigation of the Substrate and Effector Sites of Phosphoribulokinase

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Abstract
The activated spin-label 3-(2-bromoacetamido)proxyl modifies the sulfur atom of phosphorothioate-containing AMP, ADP, and ATP analogs in a facile reaction that produces a new series of spin-labeled nucleotides. One of these products, adenosine 5'-O-(S-acetamidoproxyl 3-thiotriphosphate) (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, ATP gamma SAP binds noncooperatively to the allosteric site (n = 1; KD = 8 microM). Probe bound in this site is displaced (K1/2 = 100 microM) by the allosteric effector, NADH, at concentrations comparable to those required for enzyme activation (Ka = 133 microM). In the presence of NADH, when PRK's substrate site is vacant, ATP gamma SAP binds in a cooperative mode (Hill coefficient approximately 2.9; KD = 20 microM). In the absence of NADH, ATP gamma SAP mimics ATP by exhibiting nonequilibrium binding to PRK. The observations with phosphoribulokinase, together with the straightforward nature of the methodology documented for synthesis and isolation of this class of spin-labeled nucleotides, suggest that these analogs have potentially wide application as structural probes.

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