TIME-RESOLVED LAUE STUDIES FROM DIENELACTONE HYDROLASE AND PORPHOBILINOGEN DEAMINASE. Carr P.D.¹, Robinson A.¹, Ollis D.L.¹, Hädener A.², Niemann A.C.², Helliwell J.R.³, Habash J.³, Cassetta A.³, Ursby T.⁴, Bourgois D.⁴, Schotte F⁴ and Wulff M. ⁴. ¹Res.Sch Chem., Australian National University, Canberra, Australia; ²Inst. Organic Chem., University of Basel, Switzerland; ³Dept. Struct. Chem., University of Manchester, UK; ⁴ESRF, Grenoble France

Beamline BL3 at the ESRF has been used to collect Laue diffraction patterns from crystals of the enzymes dienelactone hydrolase (DLH) and porphobilinogen deaminase (PD).

Flow cell experiments were performed on both crystal systems using substrates of varying efficacy. Time-dependent intensity changes were observed during the PD experiments that are in broad agreement with solution kinetic measurements (Neimann et al., 1994). Mutant C123S crystals of DLH were used for the flow cell experiments because previous work (Pathak & Ollis, 1990) has shown that wild type crystals are susceptible to deactivation due to oxidation of the active site residue Cys123. Disordering/reordering phenomena were observed when the substrates dienelactone and methyl dienelactone were flowed over these crystals.

In addition to the flow cell experiments data were collected on wild type DLH crystals to study the time-dependent nature of the oxidation of the active site Cys123 residue. Data were collected both on the in house CCD detector system and image plates. Initial results will be presented from these two studies.

References:

Neimann A, Matzinger P & Hädener A (1994) Helvetica Chimca Acta ,77 1791 - 1809

Pathak D & Ollis D.L. (1990) J. Mol. Biol., 214, 497 - 525