Site-specific and random immobilization of thermolysin-like proteases reflected in the thermal inactivation kinetics.

Immobilization of proteins usually leads to random orientation of the molecules on the surface of the carrier material, whereby mechanistic interpretations of changes in properties, such as thermal stability, become very difficult. Recently, we have prepared several mutant enzymes of the thermolysin-like neutral protease from Bacillus stearothermophilus, containing cysteine residues in different positions on the surface of the protein molecule. These enzymes allowed site-specific immobilization to Activated Thiol-Sepharose and showed that stabilization effects strongly depend on the position of attachment [Mansfeld, Vriend, Van den Burg, Eijsink and Ulbrich-Hofmann (1999) Biochemistry 38, 8240-8245]. The greatest stabilization was achieved after immobilization of the mutant enzymes S65C and T56C/S65C within the structural region (positions 56-69) where unfolding is initiated. In this study thermal inactivation kinetics of these two mutant enzymes, as well as those of the pseudo-wild-type enzyme and thermolysin, were compared for different types of immobilization. Besides site-specific immobilization via thiol groups, the enzymes were bound randomly via their amino groups or by mixed-type binding. The basic matrix was Sepharose 4B in all carriers. Whereas the enzymes bound site-specifically to Activated Thiol-Sepharose showed clear first-order inactivation kinetics like the soluble enzymes, the other immobilized enzyme preparations were characterized by distinct biphasic inactivation kinetics reflecting the heterogeneity of enzyme molecules on the carrier with respect to thermal unfolding. Site-specific binding resulted in stronger stabilization than the mixed binding type. However, immobilization to a highly functionalized carrier via amino groups increased stability further, suggesting that multiple fixation outside of the unfolding region 56-65 is able to increase stability of the enzyme molecules additionally.

Probing the unfolding region in a thermolysin-like protease by site-specific immobilization.

Protein stabilization by immobilization has been proposed to be most effective if the protein is attached to the carrier at that region where unfolding is initiated. To probe this hypothesis, we have studied the effects of site-specific immobilization on the thermal stability of mutants of the thermolysin-like protease from Bacillus stearothermophilus (TLP-ste). This enzyme was chosen because previous studies had revealed which parts of the molecule are likely to be involved in the early steps of thermal unfolding. Cysteine residues were introduced by site-directed mutagenesis into various positions of a cysteine-free variant of TLP-ste. The mutant enzymes were immobilized in a site-specific manner onto Activated Thiol-Sepharose. Two mutants (T56C, S65C) having their cysteine in the proposed unfolding region of TLP-ste showed a 9- and 12-fold increase in half-lives at 75 degrees C due to immobilization. The stabilization by immobilization was even larger (33-fold) for the T56C/S65C double mutant enzyme. In contrast, mutants containing cysteines in other parts of the TLP-ste molecule (N181C, S218C, T299C) showed only small increases in half-lives due to immobilization (maximum 2.5-fold). Thus, the stabilization obtained by immobilization was strongly dependent on the site of attachment. It was largest when TLP-ste was fixed to the carrier through its postulated unfolding region. The concept of the unfolding region may be of general use for the design of strategies to stabilize proteins.

The effect of hexadecylphosphocholine on the degradation of mitochondrial phospholipids.

Hexadecylphosphocholine (HePC) is known as antitumor agent but the mechanism has not yet been understood. In rat liver mitochondria its effect on phospholipid transformation has been studied by quantitative HPTLC and phosphorus determination. From the results it can be concluded that HePC influences the activities of phospholipase A2, phospholipase C, phospholipase D, and lysophospholipase A. The phospholipid transformation as well as the influence of HePC are affected by exogenous calcium ions. In the presence of calcium HePC has been found to inhibit enzyme activities, whereas in the absence of exogenous calcium ions enzymatic phospholipid transformations are activated or inhibited depending on HePC concentrations.

Extreme stabilization of a thermolysin-like protease by an engineered disulfide bond.

The thermal inactivation of broad specificity proteases such as thermolysin and subtilisin is initiated by partial unfolding processes that render the enzyme susceptible to autolysis. Previous studies have revealed that a surface-located region in the N-terminal domain of the thermolysin-like protease produced by Bacillus stearothermophilus is crucial for thermal stability. In this region a disulfide bridge between residues 8 and 60 was designed by molecular modelling, and the corresponding single and double cysteine mutants were constructed. The disulfide bridge was spontaneously formed in vivo and resulted in a drastic stabilization of the enzyme. This stabilization presents one of the very few examples of successful stabilization of a broad specificity protease by a designed disulfide bond. We propose that the success of the present stabilization strategy is the result of the localization and mutation of an area of the molecule involved in the partial unfolding processes that determine thermal stability.

Alkylphosphate esters as inhibitors of phospholipase D.

Alkylphosphate esters were shown to be potent inhibitors of phospholipase D. Using phosphatidyl choline/sodium dodecylsulfate (2:1) as substrate, IC50 values were determined for alkylphosphocholines of different chain length (C10-C18) and for various octadecylphosphate esters with different polar head groups. The inhibitory potency strongly increased with increasing chain length of the alkyl chain. The substitution of choline for heterocyclic nitrogen compounds or for 2-trimethylarsonio-ethanol also affected the inhibition of phospholipase D. Octadecylphosphocholine proved to be the most efficient inhibitor (IC50 = 6.4 microM).