Titmas R C, Angeles T S, Sugasawara R, Aman N, Darsley M J, Blackburn G, Martin M T
IGEN, Inc., Rockville, MD 20852.
Appl Biochem Biotechnol. 1994 May-Jun;47(2-3):277-90; discussion 291-2. doi: 10.1007/BF02787940.
Because there are many known C-terminally amidated peptides of biological importance, there is great potential in medicine and organic synthesis for antibodies that catalyze primary amide bond hydrolysis or formation. We characterized a catalytic antibody, 13D11, raised to a phosphinate hapten, that hydrolyzed the primary amide of a dansyl-alkylated derivative of (R)-phenylalaninamide (DNS-(R)F-NH2). At pH 9.0, 13D11 hydrolyzed DNS-(R)F-NH2 with a kcat of 1.65 x 10(-7) s-1 (kcat/kuncat = 132) and a Km of 432 microM, and was stereospecifically hapten-inhibited (Ki = 14.0 microM). Control experiments indicated that the catalytic activity was not the result of a contaminating protease. In accordance with the hapten being a transition-state analog of base hydrolysis, the rate of DNS-(R)F-NH2 hydrolysis increased with hydroxide concentration to an optimum pH of 9.5. Above pH 9.5, activity declined rapidly suggesting the antibody was inactivated during the long incubation period. This work demonstrates the feasibility of generating catalytic antibodies to hydrolyze unactivated amide bonds without cofactor assistance.
由于存在许多已知的具有生物学重要性的C末端酰胺化肽，因此对于催化伯酰胺键水解或形成的抗体而言，在医学和有机合成方面具有巨大潜力。我们对一种针对次膦酸酯半抗原产生的催化抗体13D11进行了表征，该抗体可水解（R）-苯丙氨酰胺的丹磺酰烷基化衍生物（DNS-（R）F-NH2）的伯酰胺。在pH 9.0条件下，13D11水解DNS-（R）F-NH2的kcat为1.65×10^（-7）s^-1（kcat/kuncat = 132），Km为432μM，并且具有立体特异性的半抗原抑制作用（Ki = 14.0μM）。对照实验表明，催化活性不是由污染的蛋白酶导致的。鉴于半抗原是碱水解的过渡态类似物，DNS-（R）F-NH2的水解速率随氢氧根离子浓度增加，在pH 9.5时达到最佳。在pH 9.5以上，活性迅速下降，表明抗体在长时间孵育过程中失活。这项工作证明了在没有辅因子协助的情况下产生催化抗体以水解未活化酰胺键的可行性。
