THE CATALYTIC TRIAD OF THE INFLUENZA-C VIRUS GLYCOPROTEIN HEF ESTERASE - CHARACTERIZATION BY SITE-DIRECTED MUTAGENESIS AND FUNCTIONAL-ANALYSIS

Influenza C virus is able to inactivate its own cellular receptors by virtue of a sialate 9-O-acetylesterase that releases the acetyl residu e at position C-9 of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac(2)). The receptor-destroying enzyme activity is a function of the surface glycoprotein HEF and this esterase belongs to the class of serine hydr olases. In their active site, these enzymes contain a catalytic triad made up of a serine, a histidine and an aspartic acid residue. Sequenc e comparison with other serine esterases has indicated that, in additi on to serine-71 (S71), the amino acids histidine-368 or -369 (H368/369 ) and aspartic acid 261 (D261) are the most likely candidates to form the catalytic triad of the influenza C virus glycoprotein. By site-dir ected mutagenesis, mutants were generated in which alanine substituted for either of these amino acids. Using a phagemid expression vector, pSP1D-HEF the HEF gene was expressed in both COS 7 and MDCK I cells. T he glycoprotein was obtained in a functional form only in the latter c ells, as indicated by its transport to the cell surface and measurable enzyme activity. The low level of expression could be increased by st imulating the NF-kappa B-binding activity of the cytomegalovirus immed iately promoter/enhancer element of the vector. The esterase activity of the mutant proteins was compared with that of the wild-type glycopr otein. With Neu5,9Ac(2) as the substrate, the esterase specific activi ties of the S71/A mutant and the H368,369/A mutant were reduced by mor e than 90%. In the case of the D261/A mutant the specific activity was reduced by 64%. From this data we conclude that S71, H368/369 and D26 1 are likely to represent the catalytic triad of the influenza C virus glycoprotein KEF. In addition, N280 is proposed to stabilize the oxya nion of the presumptive transition state intermediate formed by the en zyme-substrate complex.