A snapshot of enzyme catalysis using electrospray ionization mass spectrometry.
Insights into the early molecular events involving protein-ligand/substrate interactions such as protein signaling and enzyme catalysis can be obtained by examining these processes on a very short, millisecond time scale. We have used time-resolved electrospray mass spectrometry to delineate the catalytic mechanism of a key enzyme in bacterial lipopolysaccharide biosynthesis, 3-deoxy-d-manno-2-octulosonate-8-phosphate synthase (KDO8PS). Direct real-time monitoring of the catalytic reaction under single enzyme turnover conditions reveals a novel hemiketal phosphate intermediate bound to the enzyme in a noncovalent complex that establishes the reaction pathway. This study illustrates the successful application of mass spectrometry to reveal transient biochemical processes and opens a new time domain that can provide detailed structural information of short-lived protein-ligand complexes.
Li
Zhili
Z
Department of Pharmacology, Yale University School of Medicine, New Haven, CT, USA.
Sau
Apurba K
AK
Shen
Shida
S
Whitehouse
Craig
C
Baasov
Timor
T
Anderson
Karen S
KS
D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
C020011:2-dehydro-3-deoxyphosphooctonate aldolase; D000446:Aldehyde-Lyases