The Effects of Modifying the Surface Charge on the Catalytic Activity of a Thermolysin-like Protease

Abstract

The impact of long range electrostatic interactions on catalysis in the thermolysin-like protease from Bacillus stearothermophilus was studied by analyzing the effects of inserting or removing charges on the protein surface. Various mutations were introduced at six different positions, and double-mutant cycle analysis was used to study the extent to which mutational effects were interdependent. The effects of single point mutations on the k cat /K m were non-additive, even in cases where the point mutations were located 10 Å or more from the active site Zn 2؉ and separated from each other by up to 25 Å. This shows that catalysis is affected by large electrostatic networks that involve major parts of the enzyme. The interdependence of mutations at positions as much as 25 Å apart in space also indicates that other effects, such as active site dynamics, play an important role in determining active site electrostatics. Several mutations yielded a significant increase in the activity, the most active (quadruple) mutant being almost four times as active as the wild type. In some cases the shape of the pH-activity profile was changed significantly. Remarkably, large changes in the pH-optimum were not observed.The acceleration of reaction rates by enzymes is one of the essential prerequisites for life as we know it, and the multitude and diversity of enzymes shows that it should be possible to design an enzyme that will catalyze almost any reaction under almost any set of conditions. To achieve a high rate of acceleration, enzymes rely on charged groups in their active site that stabilize the transition state or function as acid or base catalysts in the reaction. The kinetic parameters of enzymes therefore display a significant pH dependence, which is determined by the pK a values of the active site groups.Catalysis depends on intricate electrostatic interactions, which may be noticeable over distances that are large compared with short range of interactions such as hydrogen bonds and hydrophobic contacts. Thus, larger parts of an enzyme may be involved in optimizing its catalytic center than previously thought. The long range character of electrostatic effects is illustrated by a, very limited, number of examples in the literature, showing that changes in surface charge at locations as far as 15 Å from a catalytic center may affect enzyme activity (1, 2). Unfortunately, electrostatic interactions are hard to handle theoretically not only because of their long range character but also because of intrinsic theoretical difficulties. For example, most electrostatic models still use a single rigid protein structure and, at most, two dielectric constants to account for all the dynamics of the protein. This clearly is an oversimplification of reality (3).We have studied the contribution of long range electrostatic interactions to catalysis by analyzing the effects of a series of charge mutations scattered over a larger part of the surface of a thermolysin-like protease from Bacillus stearothermophilus (TLP-ste).

Cloning, Biochemical Properties, and Distribution of Mycobacterial Haloalkane Dehalogenases

“…Each buffer used for characterization of DmbA had a different pH optimum. Evidence for the double pH optimum has been previously reported, for example, with potato invertase (40), 1,2-diacylglycerol kinase of the human erythrocyte membrane (1), UDP-glucose dehydrogenase (39), ATP-dependent DNase (11), and thermolysin-like protease (10). Haloalkane dehalogenases use three ionizable amino acids for their catalysis, but DmbA is currently the only family member showing more than one pH optimum.…”

Redesigning protein pKavalues

“…However, we have no data to prove that mutations at 10 Å are at a ''safe'' distance from an enzyme active site. Indeed, experimental studies (de Kreij et al 2002;Rajagopalan et al 2002) have shown that even mutations Figure 4. The average maximum abs(DpK a value) obtainable for all targets as a function of a number of mutations and minimum distance allowed between any atom of the mutated residue and any atom of the target residue.…”

Modification of enzyme surface negative charges via covalent immobilization for tailoring the activity and enantioselectivity

“…A literature survey indicated that an alteration of enzyme surface charges via site-directed mutagenesis or chemical modification was an effective way to change the active-site electrostatics, and hence the ionization constants of amino acid residues in the active-site (Afzal et al, 2007;de Kreij et al, 2002;DeSantis and Jones, 1998;Loewenthal et al, 1993). This finding has induced our hypothesis that the ionization constant of catalytic imidazolium may increase, if the hydrolase is immobilized on a hexamethylenamino-activated support (Sepabeads @ EC-HA) to decrease the negative surface charges via multipoint attachments of the carboxylic acid groups to the support.…”