Rabbit Anti-Human BPHL Polyclonal Antibody

Valacyclovir is the 5'-valyl ester prodrug of acyclovir, an effective anti-herpetic drug. Systemic availability of acyclovir in humans is three to five times higher when administered orally as the prodrug. The increased bioavailability of valacyclovir is attributed to carrier-mediated intestinal absorption, via the hPEPT1 peptide transporter, followed by the rapid and complete conversion to acyclovir. The one or more human enzymes responsible for in vivo activation of the prodrug to the active drug and its conversion sites, however, have not been identified. In this report, we describe the purification, identification, and characterization of a human enzyme that activates valacyclovir to acyclovir. A protein with significant hydrolytic activity toward valacyclovir, the 5'-glycyl ester of acyclovir, and the 5'-valyl ester of zidovudine (AZT), was purified from Caco-2 cells derived from human intestine. Using a non-redundant data base search, the N-terminal 19-amino acid sequence of the purified 27-kDa, basic protein revealed a perfect match within the N terminus of a serine hydrolase, Biphenyl hydrolase-like (BPHL, gi: 4757862) protein, previously cloned from human breast carcinoma. Recombinant BPHL exhibited significant hydrolytic activity for both valacyclovir and valganciclovir with specificity constants (k(cat)/K-m), 420 and 53.2 mM(-1).s(-1), respectively. We conclude that BPHL may be an important enzyme activating valacyclovir and valganciclovir in humans and an important new target for prodrug design.

BphI, a pyruvate-specific class H aldolase found in the polychlorinated biphenyls (PCBs) degradation pathway, catalyzes the reversible C C bond cleavage of (4S)-hydroxy-2-oxoacids to form pyruvate and an aldehyde.. Mutations were. introduced into bphI to probe the contribution of active site residues to substrate recognition and catalysis. In contrast to the wild-type enzyme that has similar specificities for acetaldehyde and propionaldehyde, the. L87A variant exhibited a 40-fold preference for propionaldehyde over acetaldehyde.,The, specificity Constant of the L89A variant in the aldol addition reaction using pentaldehyde is, increased similar to 50-fold; making it more catalytically efficient for pentaldehyde utilization compared to the wild-type utilization of the natural substrate, acetaldehyde. Replacement of Tyr-290 with phenylalanine or serine resulted in a loss of stereochemical control as the variants were able to utilize substrates with both R and S:configurations at C4 with similar kinetic parameters. Aldol cleavage and pyruvate alpha-proton exchange activity were undetectable in the R16A variant, supporting the role of Arg-16 in stabilizing a pyruvate enolate intermediate. The pH :dependence of the enzyme is consistent with a single deprotonation by a catalytic base with pK(a) values of approximately 7. In H20A, and H20S variants, pH profiles show the dependence of enzyme activity on hydroxide concentration. On the basis of these results, a catalytic mechanism is proposed.