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. 2003 Dec 15;376(Pt 3):813–821. doi: 10.1042/BJ20030716

Improvement in hydrolytic antibody activity by change in haptenic structure from phosphate to phosphonate with retention of a common leaving-group determinant: evidence for the 'flexibility' hypothesis.

Sheraz Gul 1, Sanjiv Sonkaria 1, Surapong Pinitglang 1, José Florez-Alvarez 1, Syeed Hussain 1, Emrys W Thomas 1, Elizabeth L Ostler 1, Gerard Gallacher 1, Marina Resmini 1, Keith Brocklehurst 1
PMCID: PMC1223799  PMID: 12946271

Abstract

To investigate the hypothesis that decreased hapten flexibility may lead to increased catalytic antibody activity, we used two closely related immunogens differing only in the flexibility of the atomic framework around the structural motif of the haptens, analogous to the reaction centre of the corresponding substrates. Identical leaving-group determinants in the haptens and identical leaving groups in the substrates removed the ambiguity inherent in some data reported in the literature. Anti-phosphate and anti-phosphonate kinetically homogeneous polyclonal catalytic antibody preparations were compared by using carbonate and ester substrates respectively, each containing a 4-nitrophenolate leaving group. Synthetic routes to a new phosphonate hapten and new ester substrate were developed. The kinetic advantage of the more rigid anti-phosphonate/ester system was demonstrated at pH 8.0 by a 13-fold advantage in k(cat)/k(non-cat) and a 100-fold advantage in the proficiency constant, k(cat)/k (non-cat) x K(m). Despite these differences, the pH-dependences of the kinetic and binding characteristics and the results of chemical modification studies suggest closely similar catalytic mechanisms. The possible origin of the kinetic advantage of the more rigid hapten/substrate system is discussed.

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Selected References

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