nitrophenols and 4-nitrophenyloctanoate

In this study, to explore the plasticity of the alpha/beta-hydrolase fold family, we converted bromoperoxidase A2 (BPO-A2) from Streptomyces aureofaciens to a lipase by structure comparison with lipase A (LipA) from Bacillus subtilis. These two enzymes have similar structures (2.1 A rmsd) and a very low level of sequence identity ( approximately 18%). A variant BL1 was constructed by deleting the caplike domain of BPO-A2 and further fine-tuning the newly formed substrate binding site. The lipase activity was successfully transplanted on BL1, while the halogenation activity was totally lost. BL1 also showed higher hydrolytic activities toward long chain p-nitrophenyl esters, such as p-nitrophenyl caprylate (3.7-fold) and p-nitrophenyl palmitate (7.0-fold), while its activity toward a short chain ester (p-nitrophenyl acetate) decreased dramatically, to only 1.2% of that of BPO-A2. After two rounds of directed evolution and site-directed mutagenesis on selected residues, several mutants with both improved hydrolytic activities and substrate preferences toward long chain substrates were obtained. The highest hydrolytic activity toward p-nitrophenyl palmitate of the best mutant BL1-2-E8-plusI was improved by 40-fold compared with that of BL1. These results demonstrate the possibility of manipulating the caplike domain of alpha/beta-hydrolase fold enzymes and provide further understanding of the structure-function relationship of the alpha/beta-hydrolase fold enzymes. The design strategy used in this study could serve as a useful approach for constructing variants with targeted catalytic properties using the alpha/beta-hydrolase fold.

Topics: Bacillus subtilis; Binding Sites; Biocatalysis; Caprylates; Esters; Hydrolysis; Lipase; Mutation; Nitrophenols; Palmitates; Peroxidases; Protein Conformation; Streptomyces aureofaciens; Substrate Specificity

The fatty acyl (lipid) p-nitrophenyl esters p-nitrophenyl caprylate, p-nitrophenyl laurate and p-nitrophenyl palmitate that are incorporated at a few mol % into mixed micelles with Triton X-100 are substrates for bovine milk lipoprotein lipase. When the concentration of components of the mixed micelles is approximately equal to or greater than the critical micelle concentration, time courses for lipoprotein lipase-catalyzed hydrolysis of the esters are described by the integrated form of the Michaelis-Menten equation. Least square fitting to the integrated equation therefore allows calculation of the interfacial kinetic parameters Km and Vmax from single runs. The computational methodology used to determine the interfacial kinetic parameters is described in this paper and is used to determine the intrinsic substrate fatty acyl specificity of lipoprotein lipase catalysis, which is reflected in the magnitude of kcat/Km and kcat. The results for interfacial lipoprotein lipase catalysis, along with previously determined kinetic parameters for the water-soluble esters p-nitrophenyl acetate and p-nitrophenyl butyrate, indicate that lipoprotein lipase has highest specificity for the substrates that have fatty acyl chains of intermediate length (i.e. p-nitrophenyl butyrate and p-nitrophenyl caprylate). The fatty acid products do not cause product inhibition during lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles. The effects of the nucleophiles hydroxylamine, hydrazine, and ethylenediamine on Km and Vmax for lipoprotein lipase catalyzed hydrolysis of p-nitrophenyl laurate are consistent with trapping of a lauryl-lipoprotein lipase intermediate. This mechanism is confirmed by analysis of the product lauryl hydroxamate when hydroxylamine is the nucleophile. Hence, lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles occurs via an interfacial acyl-lipoprotein lipase mechanism that is rate-limited by hydrolysis of the acyl-enzyme intermediate.

Topics: Animals; Caprylates; Cattle; Female; Kinetics; Laurates; Lipid Metabolism; Lipoprotein Lipase; Mathematics; Micelles; Milk; Nitrophenols; Palmitates