THE MECHANISM OF HYDROXYNITRILE LYASE FROM HEVEA BRASILIENSIS (HBHNL): A CATALYTIC TRIAD WITH A NON-NUCLEOPHILIC SERINE
K. M. Hainbucher, M. Gugganig, J. Zuegg, U. G. Wagner, C. Kratky
Department of Structural Biology, Institute of Physical Chemistry Karl Franzens University, Heinrichstra e 28, 8010 Graz, Austria e-mail: karl.hainbucher@kfunigraz.ac.at
Hydroxynitrile lyases (Hnl) are enzymes that catalyse the decomposition of cyanohydrins into HCN and the corresponding aldehydes or ketones. This reaction is called cyanogenesis. Its natural function in plants is the defence against predators. As cyanogenesis is known in over 3000 species, there are also different Hnls concerning physical and chemical properties like molecular mass, glycosylation, FAD dependence, etc.
Hnl from hevea brasiliensis (rubber tree) is an unglycosylated, homodimeric enzyme with a subunit molecular mass of 29.2 kDa that needs no coenzyme. Its natural substrate is acetone cyanohydrin. Yet HbHnl accepts a wide variety of cyanohydrins deriving from aliphatic or aromatic aldehydes and methyl ketones.
The reverse reaction - the formation of cyanohydrins from aldehydes and ketons - is of interest in organic synthesis as a means of forming C-C bonds. This reaction can also be performed base catalysed without an enzyme. But the great advantage in the usage of Hnl is the production of enantiomerically pure cyanohydrins in acidic solution.
The crystal structure of HbHnl was determined at our laboratory to a crystallographic resolution of 1.9 A. This enzyme turned out to have a central b-sheet surrounded by crossing a-helices, a fold known as a/b-hydrolase fold. Besides that the catalytic triad with an active nucleophile, a histidine and an acid residue is a common feature of the a/b hydrolase superfamily. For topological an structural reasons Ser80-His235-Asp207 could be identified as the catalytic triad of HbHnl. This finding could be confirmed by mutation studies. Also Cys81 proofed to be crucial for enzymatic activity.
In addition to the prototypic hydrolase fold HbHnl has an insertion which builds up a cap region. The active site with Ser80 is connected to the protein surface only through a narrow tunnel formed by the cap region. In the established reaction mechanism of serine hydrolases the nucleophile serine attacks the carbonyl carbon of the peptide (or ester) bond to form a covalently linked acyl enzyme intermediate. In analogy to this reaction a mechanism involving a hemiacetal enzyme intermediate was proposed.
There exists a group of reactants that covalently modify the nucleophilic serine in serine hydrolases and thereby inactivate the enzyme. This reaction is considered to be a diagnostic test for serine hydrolases.
We tried to inhibit HbHnl with PMSF (phenylmethylsulfonyl fluoride), DFP (diisopropyl fluorophosphate) and E 600 (paraoxon, diethyl p-nitrophenyl phosphate). While DFP and PMSF rapidly and irreversibly inhibit chymotrypsin, they have very little effect on HbHnl at a molar ratio of 10:1. Only at considerably higher concentrations (molar ratio of 10000:1) they appear to act as reversible inhibitors. As there is no irreversible inhibition, it is unlikely that a covalently linked HNL-inhibitor complex is formed. As a consequence the active Ser80 is either less nucleophilic or less accessible. We are currently performing crystallographic experiments to elucidate the nature of the interaction between Hnl and these inhibitors.