Development of an internally quenched fluorescent substrate and a continuous fluorimetric assay for Streptococcus pneumoniae signal peptidase I.
Signal peptidase (SPase) I is responsible for the cleavage of signal peptides of many secreted proteins in bacteria and serves as a potential target for the development of novel antibacterial agents due to its unique physiological and biochemical properties. In this paper, we describe a novel fluorogenic substrate, KLTFGTVK(Abz)PVQAIAGY(NO2)EWL, in which 2-aminobenzoic acid (Abz) and 3-nitrotyrosine (Y(NO2)) were used as the fluorescent donor and acceptor, respectively. The substrate can be cleaved by both Streptococcus pneumoniae and Escherichia coli SPase I. Upon cleavage of the fluorogenic substrate by SPase I, the fluorescent intensity increases and can be monitored continuously by spectrofluorometer. Kinetic analysis with S. pneumoniae SPase I demonstrated that the K(m) value for the substrate is 118.1 microM, and the k(cat) value is 0.032 s(-1). Mass spectrometric analysis and peptide sequencing of the two cleaved products confirmed that the cleavage occurs specifically at the predicted site. More interestingly, the positively charged lysine in the N-terminus of the substrate was demonstrated to be important for effective cleavage. Phospholipids were found to stimulate the cleavage reaction. This stimulation by phospholipids is dependent upon the N-terminal charge of the substrate, indicating that the interaction of the positively charged substrate with anionic phospholipids is important for maintaining the substrate in certain conformation for cleavage. The substrate and assay described here can be readily automated and utilized for the identification of potential antibacterial agents.
Peng
S B
SB
Infectious Diseases Research, Lilly Research Laboratories, Indianapolis, IN 46285, USA. Peng_Sheng_Bin@Lilly.com
Zheng
F
F
Angleton
E L
EL
Smiley
D
D
Carpenter
J
J
Scott
J E
JE
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D006090:Gram-Negative Bacteria / Q000201:enzymology; D008239:Lysine / Q000737:chemistry; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D011498:Protein Precursors; D012697:Serine Endopeptidases / Q000032:analysis* / Q000302:isolation & purification; D013296:Streptococcus pneumoniae / Q000201:enzymology*; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D008565:Membrane Proteins; D010446:Peptide Fragments; D011498:Protein Precursors; D012697:Serine Endopeptidases; C026515:type I signal peptidase; D008239:Lysine