Affinity Labeling of Nucleotide Sites in Proteins

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Colman 9728202 1. Technical Nucleotides have a multi-functional role in cellular metabolism: involved in kinase reactions, participate in dehydrogenase reactions as part of the coenzyme molecule, function as regulators of many allosteric enzymes, engaged (through the effects of cyclic AMP) in the action of several hormones, and contribute to the aggregation of platelets. This study is aimed to develop tools for a systematic exploration of the amino acid residues in the nucleotide sites of proteins. The general approach of affinity is used, with reagents previously synthesized in this laboratory and new reagents which incorporate, at various positions of the purine or ribose ring alkylating agents capable of reacting covalently with the protein; the nucleotide moiety thus provides the specificity for chemical modification of the proteins within the binding sites. The effects of these nucleotide alkylating agents are examined on several enzymes; adenylosuccinate synthetase and adenylosuccinate lyase, which catalyze successive reactions in the conversion of inosine monophosphate to AMP; and cyclic AMP phosphodiesterases, which catalyzes the hydrolysis of cAMP to form 5' - AMP. For each protein, the kinetics of the affinity labeling reaction is examined, the extent of reagent incorporation determined and the radioactive peptides isolated from proteolytic digests of labeled enzyme and identified with the modified residue. The catalytic and ligand binding properties as well as the conformational characteristics of the modified enzymes are also evaluated. For most of these enzymes, the results of these enzymes in solution are compared with their determined crystal structures. The nucleotide analogs, fluorosulfonylbenzoyl nucleosides, have been tested on more than 50 proteins; with results promising for its general applicability to other enzymes. They are useful for the detection of nucleotide binding proteins expressed by cloning. The significance of this may rest with providing the k nowledge base for rational design of specific inhibitors targeted to particular nucleotide-requiring enzymes. 2. Non-technical Nucleotides have a multifunctional role in cellular metabolism, such as participating in enzymatic reactions, activating regulatory enzymes, and serving as "second messenger" like cyclic AMP. This study is aimed to develop the tools which allow a systematic exploration of the enzyme which binds nucleotides. A general approach of "affinity labeling" is used to attach a nucleotide moiety of the new reagent compound to a specific binding site on the protein, irreversibly, so that the binding site can be isolated and characterized. The effects of nucleotide alkylating agents on two purine nucleotide biosynthesis enzymes and on an enzyme important in the degradation of nucleotide cyclic AMP are studied. Nucleotide analogues such as fluorsulfonylbenzoyl nucleosides which react to proteins at specific sites are specifically probed. For biotechnology, these molecules can be used to detect expression of cloned proteins, and to provide the knowledge base for rational design of inhibitors for nucleotide-requiring enzymes.