UNSPECIFIED (1997) Inactivation of the regulatory protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath) by proteolysis can be overcome by a Gly to Gin modification. EUROPEAN JOURNAL OF BIOCHEMISTRY, 248 (1). pp. 72-79. ISSN 0014-2956

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Abstract

The regulatory protein B of soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), exists as a mixture of the full-length active form and truncated farms, B' and B ''. Electrospray ionisation mass spectrometry (ESI-MS) was used to identify a cleavage site between Met12 and Gly13, such that 12 amino acids were lost from the N-terminus of protein B. This truncate was designated B' and molecular masses were assigned to proteins B and B' of 15852.6 +/- 0.4 Da and 14629.5 +/- 0.3 Da, respectively. A cleavage site between Gln29 and Val30 was also identified such that 29 amino acids were lost from the N-terminus of protein B. This truncate was designated B '' and had a molecular mass of 12709.93 +/- 0.02 Da. Proteins B' and B '' were found to be inactive in the sMMO system. Addition of protease inhibitors or the heterologous expression of protein B in various strains of lon-deficient or ompT-deficient Escherichia coli, did not inhibit B' formation. Expression of protein B as a glutathione S-transferase fusion protein and subsequent purification of protein B from a coli using affinity chromatography resulted in preparations of protein B with higher enzyme activities than that of wild-type protein B. However, ESI-MS confirmed that protein B' was still present. Alteration of the Met12-Gly13 cleavage site to Met12-Gln13 revealed that the stability of G13Q at 20 degrees C and 37 degrees C was higher than that of wild-type preparations. ESI-MS indicated that protein B' was absent and could only be identified after prolonged incubation at room temperature. The amount of active protein B present in the cell may be controlled by protein B cleavage, thereby regulating electron transfer. Alternatively, it may allow protein B to maintain a certain conformation necessary for enzyme activity and this may control the activity of sMMO in response to the supply of methane to the cell.

Item Type:	Journal Article
Subjects:	Q Science > QD Chemistry
Journal or Publication Title:	EUROPEAN JOURNAL OF BIOCHEMISTRY
Publisher:	SPRINGER VERLAG
ISSN:	0014-2956
Date:	15 August 1997
Volume:	248
Number:	1
Number of Pages:	8
Page Range:	pp. 72-79
Publication Status:	Published
URI:	http://wrap.warwick.ac.uk/id/eprint/16476

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