Engineered Metal Regulation of Trypsin Specificity
Biochemistry 1995, 34, 2172-2180, 10.1021/bi00007a010
Histidine substrate specificity has been engineered into trypsin by creating metal binding sites for Ni2+ and Zn2+ ions. The sites bridge the substrate and enzyme on the leaving-group side of the scissile bond. Application of simple steric and geometric criteria to a crystallographically derived enzyme- substrate model suggested that histidine specificity at the P2' position might be acheived by a tridentate site involving amino acid residues 143 and 151 of trypsin. Trypsin N143H/E151H hydrolyzes a P2'- His-containing peptide (AGPYAHSS) exclusively in the presence of nickel or zinc with a high level of catalytic efficiency. Since cleavage following the tyrosine residue is normally highly disfavored by trypsin, this result demonstrates that a metal cofactor can be used to modulate specificity in a designed fashion. The same geometric criteria applied in the primary SI binding pocket suggested that the single-site mutation D189H might effect metal-dependent His specificity in trypsin. However, kinetic and crystallographic analysis of this variant showed that the design was unsuccessful because His 189 rotates away from substrate causing a large perturbation in adjacent surface loops. This observation suggests that the reason specificity modification at the trypsin S1 site requires extensive mutagenesis is because the pocket cannot deform locally to accommodate alternate PI side chains. By taking advantage of the extended subsites, an alternate substrate specificity has been engineered into trypsin.
Metal: ZnLigand type: Amino acidHost protein: TrypsinAnchoring strategy: DativeOptimization: GeneticReaction: Hydrolytic cleavageMax TON: ---ee: ---PDB: ---Notes: Substrate specificty
Metal: NiLigand type: Amino acidHost protein: TrypsinAnchoring strategy: DativeOptimization: GeneticReaction: Hydrolytic cleavageMax TON: ---ee: ---PDB: ---Notes: Substrate specificty
Thermostable Peroxidase-Activity with a Recombinant Antibody L-Chain-Porphyrin Fe(III) Complex
FEBS Lett. 1995, 375, 273-276, 10.1016/0014-5793(95)01224-3
In order to engineer a new type of catalytic antibody, we attempt to use a monoclonal antibody L chain as a host protein for a porphyrin. TCPP (meso‐tetrakis(4‐carboxyphenyl)porphyine) was chemically synthesized and Balb/c mice were immunized using TCPP as a hapten. Two hybridoma cells (03‐1, 13‐1), that produce monoclonal antibody against TCPP, were obtained. Genes for both H and L chains of monoclonal antibodies were cloned, sequenced and overexpressed using E. coli as a host. ELISA and fluorescence quenching method show that the independent antibody L chains from both Mab03‐1 and Mab13‐1 have specific interaction with TCPP. Furthermore, the recombinant antibody L chain from Mab13‐1 exhibits much higher peroxidase activity than TCPP Fe(III) alone. The enzyme activity was detectable with pyrogallol and ABTS (2,2‐azinobis‐3‐ethylbenzthiazolin‐6‐sulfonic acid) but not with catechol. This new catalytic antibody was extremely thermostable. Optimum temperature of the peroxidase reaction by the complex of 13‐1L chain and TCPP Fe(III) was 90°C, while that the TCPP Fe(III) alone was 60°C.
Metal: FeLigand type: PorphyrinHost protein: Antibody L-chain from Mab13-1 hybridoma cellsAnchoring strategy: AntibodyOptimization: ---Reaction: PeroxidationMax TON: ---ee: ---PDB: ---Notes: ---