The structure of neutral protease from <i>Bacillus cereus</i> at 0.2‐nm resolution

Stark, Wilhelm; Pauptit, Richard A.; Wilson, K.S.; Jansonius, Johan N.

doi:10.1111/j.1432-1033.1992.tb17109.x

Cited by 78 publications

(50 citation statements)

References 49 publications

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“…The amino acid sequences of several TLPs have been determined (see Ref. 4, or the Merops data base at www.merops.co.uk/merops/famcards/m4.htm), and the three-dimensional structures of TLPs isolated from several bacteria (Bacillus thermoproteolyticus (5), Bacillus cereus (6), Pseudomonas aeruginosa (7), and Staphylococcus aureus (8)) have been solved. TLPs consist of an ␣-helical C-terminal domain and a ␤-rich N-terminal domain.…”

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confidence: 99%

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

Kreij

Burg

Venema

et al. 2002

Journal of Biological Chemistry

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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).

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confidence: 99%

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

Kreij

Burg

Venema

et al. 2002

Journal of Biological Chemistry

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“…The crystal structures of thermolysin (Matthews et al, 1972;Holmes and Matthews, 1982) and of the NP from Bacillus cereus (Pauptit et al, 1988;Stark et al, 1992) have been elucidated, and on the basis of these structures three-dimensional models of other NP have been built (Signor et al, 1990;Vriend and Eijsink, 1993). Within the family of NP large differences in thermal stability occur and therefore these enzymes form an interesting system to study structurestability relationships.…”

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Protein stabilization by hydrophobic interactions at the surface

Burg

Dijkstra

Vriend³

et al. 1994

European Journal of Biochemistry

103

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The contribution of the solvent‐exposed residue 63 to thermal stability of the thermolysin‐like neutral protease of Bacillus stearothermophilus was studied by analyzing the effect of twelve different amino acid substitutions at this position. The thermal stability of the enzyme was increased considerably by introducing Arg, Lys or bulky hydrophobic amino acids. In general, the effects of the mutations showed that hydrophobic contacts in this surface‐located region of the protein are a major determinant of thermal stability. This observation contrasts with general concepts concerning the contribution of surface‐located residues and surface hydrophobicity to protein stability and indicates new ways for protein stabilization by site‐directed mutagenesis.

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“…The coordination of the zinc atom in the active site has been resolved in four metalloproteases so far, including thermolysin from Bacillus thermoproteolytics, a neutral protease from B. cereus, an elastase from Pseudomonas aeruginosa, and astacin from the crayfish, Astacus fluviatflis [1][2][3][4]. The three bacterial enzymes are closely related and contain three amino acid residues and one water molecule which bind zinc.…”

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confidence: 99%

Families of metalloendopeptidases and their relationships

1992

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Crystal structures available for four metalloendopeptidascs have revealed zinc ligands for these enzymes. New sequence information has made it possible to compare the primary structures of the zinc-binding site in metalloendapeptidases. A scheme based on the zinc-bindin8 sit~ is proposed to classify metalloendopeptidases into five distinct families: thermolysin, astacin, serratia, matrixin, and snake venom metalloproteinases. Two histidlnes and one 81utamate are zinc-ligands in the thermolysin family. Three histiciincs and one tyrosine are zinc ligands in the other four families, which are further distinguished by the identity of the residue following the third histidine and by the environment surrounding the tyro~iae.

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The structure of neutral protease from Bacillus cereus at 0.2‐nm resolution

Cited by 78 publications

References 49 publications

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

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

Protein stabilization by hydrophobic interactions at the surface

Families of metalloendopeptidases and their relationships

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