Metalloendopeptidase QG. Isolation from Escherichia coli and characterization.
A new proteinase, which preferentially cleaves the Gln-Gly bond, was isolated from Escherichia coli. Because of this narrow specificity, the enzyme was called metalloendopeptidase QG. The proteinase is a monomer and consists of a single polypeptide chain of Mr 67,000, which is significantly smaller than the other known metalloendopeptidases of E. coli. It is found in the cytoplasm, but not in the periplasm. The enzyme cleaves the substrate benzyloxycarbonyl-Gln-Gly-Pro 2-naphthylamide between the glutamine and glycine residues, as well as its extended homologues including a nonapeptide, but it does not hydrolyse either the oxidized A and B chains of insulin or azo-casein. The pH-dependence of substrate hydrolysis gives a bell-shaped curve with pK1 = 6.6 and pK2 = 8.8. The metallopeptidase is inhibited in Tris and imidazole buffers, the basic components of which are presumably liganded to the essential Zn2+ ion. 2-Aminobenzoyl-Gln-Gly-Pro 2-naphthylamide, designed as a fluorescent substrate for the metallopeptidase, proved to be a strong inhibitor. Bestatin, an inhibitor of aminopeptidases in the micromolar concentration range, inhibits the metalloendopeptidase only in the millimolar concentration range. Captopril, the widely used inhibitor of angiotensin-converting enzyme, is a fairly good inhibitor of the metalloendopeptidase. The simplest inhibitor that can be used to protect recombinant proteins from degradation by the metalloendopeptidase may be EDTA, which is effective at low millimolar concentration.
Polgár
L
L
Institute of Enzymology Biological Research Center, Hungarian Academy of Sciences, Budapest.
Szigetvári
A
A
Löw
M
M
Kóródi
I
I
Balla
E
E
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008666:Metalloendopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
D009842:Oligopeptides; D011480:Protease Inhibitors; D008666:Metalloendopeptidases; C067450:metalloendopeptidase QG