Catalytic Antibodies by Fluorescence Screening

A structural basis for the activity of retro-Diels–Alder catalytic antibodies: Evidence for a catalytic aromatic residue

“…A similar catalytic role of hydrogen bonds has been argued for Diels-Alderase antibodies 1E9 (11) and 39A11 (12). In the slower reacting substrate 1b, the H-bonding interaction would occur with the nonbonding electron pair of the bridge's NH group, which should rather slow down the retro-Diels-Alder reaction if one considers that the retro-Diels-Alder reaction does not occur in the protonated forms of the substrates (16,17). Therefore, the participation of these hydrogen bonds could at most explain the 10-fold reactivity difference between the regioisomeric substrates 1a and 1b.…”

“…Aromatic residues have been observed recurrently in the binding pockets of Diels-Alderase antibodies. We have used x-ray crystallography to identify a similar aromatic residue in the binding pocket of the retro-Diels-Alder catalytic antibodies 10F11 (16) and 9D9 (17). This aromatic residue is conserved as well in the third retro-Diels-Alder antibody 27C5.…”

Expression Improvement and Mechanistic Study of the Retro-Diels-Alderase Catalytic Antibody 10F11 by Site-directed Mutagenesis

“…6 -8 In our study of similar pericyclic processes, we have reported that catalytic antibodies 9D9 and 10F11, raised against the acridine haptens 3a -c, catalyze the retro-Diels-Alder reaction releasing nitroxyl (HNO) as a heterodienophile from the inert prodrug 1 (Figure 1). 9,10 Nitroxyl can in turn be oxidized to nitric oxide (NO) by superoxide dismutase (SOD). 11 This prodrug release system is of particular biological significance, since nitric oxide acts as a chemical messenger for a number of fundamental bioregulatory processes, including blood pressure regulation and memory.…”

“…a K TS ¼ K M =ðk cat =k uncat Þ is the transition state pseudo-dissociation constant, which measured how tightly the transition state is bound.b Relative activity as measured by the specificity constant k cat =K M . c Parameters for 9D9 and 10F11 from hybridoma by Bensel et al10 d For trace activities the determination of k cat and K M is uncertain, only the specificity constant k cat =K M and hence K TS is given. 8C for two days before recording the kinetic data.…”

Enantioselective Fluorogenic Assay of Acetate Hydrolysis for Detecting Lipase Catalytic Antibodies

“…We have recently shown that fluorogenic substrates are optimally suited for detecting catalytic antibodies directly in cell culture [6]. However, fluorogenic or chromogenic substrates for esterase-activity screening are normally esters of aromatic phenols [7].…”