Use of Transglutaminase. Reversible Blocking of Amino Groups in Substrate Proteins for a High Yield of Specific Products

Abstract

Transglutaminase catalyzes an acyl-transfer reaction in which the y-carboxyamide groups of pep tide-bound glutaminyl residues are the acyl donors. Primary amino groups in a variety of compounds act as acyl acceptors with the subsequent formation of the monosubstituted γ-amide of the peptide-bound glutamic acid. When one wants to bind a desired amino compound into glutamine residues in a protein using this enzyme, concurrent generation of intra- and intermolecular ∊-(γ-glutamyl)lysine crosslinks of the protein lowers the binding efficiency because the reactive glutamine residues are consumed by the crosslinking. Maleylation of amino groups in proteins was attempted with maleic anhydride in order to suppress this unfavorable crosslink formation. Maleylation of caseins, mainly α_s1-casein, increased the rate and the extent of transglutaminase-catalyzed incorporation of amino compounds such as putrescine, methionine ethyl ester, and NAD⁺ analog into the modified proteins. Generation of intermolecular crosslinks during the incorporation reactions was completely repressed by maleylation. Bovine serum albumin and ovalbumin are non-reactive for transglutaminase, i.e.both lysine and glutamine residues in these proteins are unavailable to this enzyme. Maleylation of both proteins induced a conformational change by which glutamine residues were exposed to be available as acceptors in the transglutaminase-catalyzed incorporation of amino compounds. Maleyl residues in proteins can be readily removed by treating the modified proteins at acidic pH. Thus maleylation improves substrate proteins for preparing a specified product with a high yield using transglutaminase.