Transglutaminase-catalysed Formation of Coenzymatically Active NAD⁺ Analog • Casein Conjugates

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An NAD⁺ analog, N⁶-[(6-aminohexyl)carbamoylmethyl]-NAD⁺, was immobilized on bovine caseins by the action of transglutaminase. Binding of NAD⁺ was not observed. It appears that NAD⁺ analog binds with α_s1- and β-caseins through formation of the γ-glutamylamine bond between the amino groups attached to the hexyl chain in NAD⁺ analog and the glutaminyl residues in caseins. The NAD⁺ analog immobilized on the caseins was enzymatically reducible by alcohol dehydrogenase; the numbers of Coenzymatically active NAD⁺ analog molecules were 0.4, 0.2, 0.9, and 0.3 per mol of α_s1-, dephosphorylated α_s1-, acetylated α_s1-, and β-caseins, respectively. The number of immobilized NAD⁺ analog molecules was always larger than that of the enzymatically reducible one, indicating that some of the immobilized NAD⁺ was inactive. Michaelis constants of immobilized NAD⁺ analog in alcohol dehydrogenase reaction were similar to those of free forms of NAD⁺ and NAD⁺ analog. Maximum velocities were reduced to 15-30% of the free NAD⁺ analog. Immobilized NAD⁺ analog was much more stable at alkaline pH than free NAD⁺ and its analog. The coenzyme • casein conjugates were recovered almost completely in casein precipitated by calcium.