Spectral Observation of an Acyl-Enzyme Intermediate of Lipoprotein Lipase

Camilo Rojas, Huei Hsiang Wang, Chris R. Lively, William G. Gustafson, Leslie O. Schulz, James T. McFarland

Research output: Contribution to journalArticlepeer-review

7 Scopus citations


There have been several studies indicating that hydrolysis reactions of fatty acid esters catalyzed by lipases proceed through an acyl-enzyme intermediate typical of serine proteases. In particular, one careful kinetic study with the physiologically important enzyme lipoprotein lipase (LPL) is consistent with r0ate-limiting deacylation of such an intermediate. To observe the spectrum of acyl-enzyme and study the mechanism of LPL-catalyzed hydrolysis of substrate, we have used a variety of furylacryloyl substrates including l,2-dipalmitoyl-3-[(β-2-furylacryloyl)triacyl]glyceride (DPFATG) to study the intermediates formed during the hydrolysis reaction catalyzed by the enzyme. After isolation and characterization of the molecular weight of adipose LPL, we determined its extinction coefficient at 280 nm to quantitate the formation of any acyl-enzyme intermediate formed during substrate hydrolysis. We observed an intermediate at low pH during the enzyme-catalyzed hydrolysis of (furylacryloyl)imidazole. This intermediate builds early in the reaction when a substantial amount of substrate has hydrolyzed but no product, furylacrylate, has been formed. The acyl-enzyme has a λmax=305 nm and a molar extinction coefficient of 22600 M−1 cm−1; these parameters are similar to those for furylacryloyl esters including the serine ester. These data provide the first spectral evidence for a serine acyl-enzyme in lipase-catalyzed reactions. The LPL hydrolysis reaction is base catalyzed, exhibiting two pKa values; the more acidic of these is 6.5, consistent with base catalysis by histidine. The biphasic rates for substrate disappearance or product appearance and the absence of leaving group effect indicate that deacylation of intermediate is rate limiting.

Original languageEnglish (US)
Pages (from-to)4475-4481
Number of pages7
Issue number10
StatePublished - 1989
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry


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