Conformational changes and loose packing promote E. coli Tryptophanase cold lability

We studied the reversible cold lability of E. coli Trpase and its Y74F, C298S and W330F mutants. In contrast to the holo E. coli Trpase all apo forms of Trpase dissociated into dimers already at 25°C and even further upon cooling to 2°C. The crystal structures of the two mutants, Y74F and C298S in their apo form were determined at 1.9？ resolution. These apo mutants were found in an open conformation compared to the closed conformation found for P. vulgaris in its holo form. This conformational change is further supported by a high pressure study.We suggest that cold lability of E. coli Trpases is primarily affected by PLP release. The enhanced loss of activity of the three mutants is presumably due to the reduced size of the side chain of the amino acids. This prevents the tight assembly of the active tetramer, making it more susceptible to the cold driven changes in hydrophobic interactions which facilitate PLP release. The hydrophobic interactions along the non catalytic interface overshadow the effect of point mutations and may account for the differences in the dissociation of E. coli Trpase to dimers and P. vulgaris Trpase to monomers.Enzymes can undergo a reversible loss of activity at low temperatures, a process that is termed cold inactivation [1,2]. This phenomenon is found in a widespread variety of oligomeric enzymes, e.g, pyrophosphatase [3], pyruvate carboxylase [4], alcohol dehydrogenase [5], as well as PLP-dependent enzymes such as glutamic acid decarboxylase [6] and tryptophanase (Trpase) [7-9]. It has been proposed that cooling evokes major changes in hydrophobic interactions which leads to destabilization of the enzyme quaternary structure concomitant with dissociation into its corresponding subunits [10]. These changes can in most cases be reversed after re-warming [11,12]. Sometimes, however, cold-induced dissociation is followed by an aggregation step, which causes the irreversibility of the process [13-16]. It is believed that at low temperatur