Affinity label periodate-oxidized nucleotides

In many of the examples cited of affinity labeling of enzymes by periodate-oxidized nucleotides, it has been assumed that the reaction involved formation of a Schiff base with an enzymic lysine, as in Fig. 3a,b. However, in very few papers has direct evidence been presented supporting the existence of a Schiff base intermediate. Lowe and Beechey (83), after examining in detail the structure of periodate-oxidized ATP, concluded that in aqueous solution there is little free aldehyde rather, the compound exists predominantly as an equilibrium mixture of three dialdehyde monohydrates (cyclic hemiacetals) and a dihydrate. The presence of cyclic hemiacetals may account for the ability of periodate-oxidized NADP and NADPH to function as coenzymes in several enzymic reactions (e.g., 78, 81). In many cases, the product of the covalent reaction of an enzyme and periodate-oxidized nucleotide may be a dihydroxymorpholino derivative (Fig. 3c), which is similar to the cyclic hemiacetals observed in aqueous solu-... 

A peptide from pyruvate kinase labeled with oADP (55) and one from ferre-doxin-NADP reductase labeled with oNADP (75) have been isolated and characterized. These are exceptions. Despite the large number of papers describing the kinetics of affinity labeling by periodate-oxidized nucleotides, there are very few reports of the identification of particular amino acids labeled by these reagents within determined peptide sequences. For enzyme products other than a Schiff base reducible by NaBHU, the instability of the products in the proteolytic digests of modified enzymes under conditions of peptide purification has precluded isolation of labeled peptides in most cases. 

As a class of affinity labels, the nucleoside polyphosphopyridoxals exhibit many of the favorable features without many of the drawbacks of the periodate-oxidized nucleotides. The nucleoside polyphosphopyridoxals are structurally close to the natural coenzymes, are water soluble and negatively charged at neutral pH, and exhibit high affinity for the enzymes with which they have been tested. Their targets have thus far been limited to lysine residues, but they react to form Schiff bases which can be stabilized by reduction with NaBH4. Once reduced, the products are stable throughout the peptide purification procedures, and the sequences of the modified peptides can readily be determined. 

Two aldehydic nucleotide derivatives have found use as affinity labels. The magnesium salt of (64), formed by oxidation of ATP with periodate, is a competitive inhibitor of pyruvate carboxylase with respect to [Mg. ATP2-],100 and (65), obtained from the / -anomer of 5-formyluridine-5 -triphosphate on treatment with alkali, is a non-competitive and reversible inhibitor of DNA-dependent RNA polymerase from E. coli.101 In each case, addition of borohydride gives stoicheiometric covalent linkage of the nucleotide to the enzyme, with irreversible inactivation. It is thought that condensation with lysine occurs to give a Schiff s base intermediate, which undergoes subsequent reduction. 

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