Mutation analysis of the histidine residues in the glycylglycine endopeptidase ALE-1.
A novel staphylolytic enzyme, ALE-1, is a glycylglycine endopeptidase produced by Staphylococcus capitis EPK1. ALE-1 possesses seven histidines. Chemical modification studies using diethylpyrocarbonate and iodoacetic acid suggested that a histidine or tyrosine residue(s) in the molecule is important for the organism's staphylolytic activity. All of the histidine residues, one tyrosine, and one aspartic acid residue in the N-terminally truncated ALE-1 (DeltaN-term ALE-1) were systematically altered by site-directed mutagenesis, and the enzyme activities and metal contents of the variants were measured. Our studies indicated that His-150, His-200, His-231, His-233, and Asp-154 are essential for the enzyme activity of DeltaN-term ALE-1. Except for His-150 and Asp-154, all of these amino acids were located within the 38-amino-acid region conserved among 11 proteins, including 5 staphylolytic endopeptidases. Inductively coupled plasma-mass spectrometric analysis of DeltaN-term ALE-1 revealed that it contains one atom of zinc per molecule. Measurement of the zinc content of the mutant DeltaN-term ALE-1 suggested that His-150 and -233 are important for zinc binding; their loss in these variant enzymes coincided with the loss of staphylolytic activity. These results strongly suggest that ALE-1 is a novel member of zinc metalloproteases.
Fujiwara
Tamaki
T
Department of Bacteriology, Hiroshima University Graduate School of Biomedical Sciences, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553, Japan.
Aoki
Shin
S
Komatsuzawa
Hitoshi
H
Nishida
Tetsuya
T
Ohara
Masaru
M
Suginaka
Hidekazu
H
Sugai
Motoyuki
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000235:genetics / Q000502:physiology; D017124:Conserved Sequence; D010450:Endopeptidases / Q000235:genetics / Q000502:physiology; D006639:Histidine / Q000235:genetics / Q000502:physiology*; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D013210:Staphylococcus / Q000201:enzymology*; D014443:Tyrosine / Q000235:genetics / Q000502:physiology; D015032:Zinc / Q000032:analysis
D001224:Aspartic Acid; D014443:Tyrosine; D006639:Histidine; D010450:Endopeptidases; D008666:Metalloendopeptidases; C104239:glycylglycyl endopeptidase ALE-1; D015032:Zinc