Karen A. Bostian

Endoproteinase Activity of Type A Botulinum Neurotoxin: Substrate Requirements and Activation by Serum Albumin

Type A botulinum neurotoxin, a zinc-dependent endoproteinase that selectively cleaves the neuronal protein SNAP-25, can also cleave relatively short peptides. We found that bovine and other serum albumins stimulated the type A-catalyzed hydrolysis of synthetic peptide substrates, through a direct effect on the kinetic constants of the reaction. Furthermore, with bovine serum albumin in the assays, the optimum substrate size was 16 residues (11 on the amino-terminal side of the cleavage site and 5 on the carboxy-terminal side). To further investigate the catalytic requirements of the neurotoxin, peptides were synthesized with various amino acid substitutions at the P5 through P5′ substrate sites. Changes at all of these locations affected values for both kcat and Km. Substitutions at the P2, P1′, and P2′ sites had more pronounced effects on hydrolysis rates than did substitutions at the P1 site. Enzyme–substrate interactions at the P3′ threonine probably involved the side-chain methyl group rather than the hydroxyl group. Replacing the P2′ alanine with leucine eliminated detectable hydrolysis, but not binding, since this peptide was an inhibitor. A negatively charged residue was preferred at P5, but not at P4. The data indicate that type A botulinum neurotoxin has an extended substrate recognition region and a requirement for arginine as the P1′ residue.

Proteolysis of synthetic peptides by type A botulinum neurotoxin.

Type A botulinum neurotoxin catalyzed the hydrolysis of synthetic peptides based on the sequence of the 25-kD synaptosomal protein SNAP-25. In each peptide, the toxin cleaved at a single glutaminyl-arginine bond corresponding to residues 197 and 198 of SNAP-25, confirming earlier reports on the enzymatic specificity of the toxin in synaptosomal preparations. Metal chelators inhibited catalysis, consistent with a metalloprotease activity. In contrast to tetanus toxin and other botulinum toxin serotypes, type A toxin hydrolyzed relatively short, 17-to 20-residue peptides. In the substrates, SNAP-25 residue 202 and one or more of residues 187–191 were required for efficient hydrolysis, but residues 167–186 and 203–206 were not. The highest rates of hydrolysis were found when the C-terminal residues of the peptides were amidated.

Type A botulinum neurotoxin proteolytic activity: development of competitive inhibitors and implications for substrate specificity at the S1' binding subsite.

Type A botulinum neurotoxin (botox A) is a zinc metalloprotease that cleaves only one peptide bond in the synaptosomal protein, SNAP‐25. Single‐residue changes in a 17‐residue substrate peptide were used to develop the first specific, competitive inhibitors of its proteolytic activity. Substrate analog peptides with P4, P3, P2′ or P3′ cysteine were readily hydrolyzed by the toxin, but those with P1 or P2 cysteine were not cleaved and were inhibitors. Peptides with either d‐ or l‐cysteine as the N‐terminus, followed by the last six residues of the substrate, were the most effective inhibitors, each with a K i value of 2 μM. Elimination of the cysteine sulfhydryl group yielded much less effective inhibitors, suggesting that inhibition was primarily due to binding of the active‐site zinc by the sulfhydryl group. Botox A displayed an unusual requirement for arginine as the P1′ inhibitor residue, demonstrating that the S1′ binding subsite of botox A is dissimilar to those of most other zinc metalloproteases. This characteristic is an important element in shaping the substrate specificity of botox A.

Protein-sparing therapy during pneumococcal infection in rhesus monkeys.

A model was developed in the rhesus monkey to determine if the marked wasting of body proteins associated with sepsis could be prevented by an intravenous supply of various nutritional substrates. All monkeys were given a basic infusion of 0.5 gm of amino acid nitrogen/kg body weight via an indwelling catheter in the jugular vein. Three groups were given diets with no added calories, 85 calories/kg from dextrose or 85 calories from lipid. In each group, six monkeys were inoculated with 3 x 10(8) Streptococcus pneumoniae and four with heatkilled organisms. In the monkeys infused with the amino acids alone, pneumococcal sepsis resulted in a fourfold increase in loss of body proteins compared with calorie-restricted controls. Addition of 85 calories/kg/day of either dextrose or lipid reduced body wasting associated with infectious disease. The calories from lipid were utilized bythe septic host as a source of energy, with a slightly reduced efficiency when compared with the isocaloric infusion of dextrose. The nitrogen sparing of the fat emulsion could not be accounted for by its glycerol content. Therefore, the septic monkey seemed to utilize fatty acids as an energy substrate. It appears that the carbohydrate calories tend to favor the synthesis of peripheral proteins (associated mainly with skeletal muscle), while lipid calories favor synthesis of visceral proteins such as plasma albumin and acute-phase proteins.