Reversible Denaturation and Reactivation of Aspartase from Hafnia alvei

Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) catalyzes the reversible conversion of L-aspasteate to fumarate and ammonium ion. Aspartase is a catabolic enzyme in both bacteria and plants, but the reaction is reversible and favors aspartate formation with ΔG = 3.2 kcal/mol for aspartate deamination. The aspartase from Hafnia alvei, that is important for aspartate synthesis in industrial purpose, is a tetramer composed of four apparently identical subunits of molecular weight 55,000 Da. Although many aspects of H. alvei aspatase reactions are well studied, the role of subunit-subunit interaction in this oligomeric protein is not known yet. As the relationship between subunit assembly and function are very important for elucidation of the protein functions at molecular basis, we have studied the activitysubunit interaction relationship and effect of environmental factors, such as temperature and pH, on reversible denaturation and reactivation of the H. alvei aspartase. As shown in Figure 1(a) at 1 M Gu-HCl was 40%. On a temperature scale (0-40 C), the optimum temperature for reactivation was attained at 25 C (Figure 2a). There was no reactivation between 0-10 C, progressed with linearly increase at 10-25 C, with a level off between 25-35 C and completely stopped at 35 C. The optimum pH for reactivation was 7.0 and little degree of reactivation was attained at the acidic or alkaline pH, presumably due to charge interaction of polypeptide chains (Figure 2b). When the temperature of renaturation was shifted from 4 to 25 C, a rapid increase in the rate of reactivation was observed with ~45 min lag phase (Figure 3), reasoning need of time for enzyme to remove the Gu-HCl by dilution. These results attributed a temperature-dependent process, as a rate-determining step in the reactivation of H. alvei aspartase. This indicates that the enzyme reached to restructured form during renaturation process and reactivated at the suitable temperature. Many cases are known, actually in which enzymes are not able to be readily reactivated simply by removal of the causes due to partial or serious denaturation of the polypeptides. However, the fact that the native confirmation of most proteins is uniquely determined by the amino acid sequence in the protein, any protein to which this generalization applies should always be able to recover its native conformation, provided that its primary sequence remains intact. Sachiko et al. identified an active dimeric form of aspartase as a denaturation intermediate of E. coli. We have proposed the intermediate forms of H. alvei aspartase during reversible denaturation (Scheme 1). As at ≥ 1 M GuHCl, aspartase dissociates to monomers and largely unfolds, in the present investigation, the denaturation process at < 1 M Gu-HCl can be explained in terms of a two-state transition. The 100% renaturation below 1 M Gu-HCl knowed out