Enzymes

Of the many thousands of enzymes that occur in nature, only a tiny fraction are irreparably damaged by the activity of their own catalytic center. Many of these are members of the eicosanoid biosynthetic pathways. Within this unique group, each enzyme has its own special characteristics that affect proper isolation and storage. LTA4 hydrolase, the enzyme which catalyzes the production of Leukotriene B4, is a good example. In addition to undergoing autoinactivation during catalytic turnover, this enzyme has amino peptidase activity permitting it to degrade itself proteolytically in the absence of substrate. Or consider Prostaglandin H Synthase 2, which is so unstable to biochemical manipulation that a significant portion of the enzyme is lost during purification. We provide these special enzymes along with a carefully detailed description of proper storage, handling and activity.

Prostaglandin H Synthase (PGHS), also known as cyclooxygenase, is our most important enzyme product. PGHS catalyzes the conversion of arachidonic acid to Prostaglandin H2. The fact that Prostaglandin H2 serves as an intermediate in the biosynthesis of prostaglandins and thromboxanes makes PGHS an important target for anti-inflammatory drug development.

For over two decades only one form of this enzyme was identified and isolated from a variety of tissues. Although several reports appeared in the literature suggesting a second form of PGHS, confirmation of its existence came in 1991 when Simmons (Brigham Young University, Provo) and Herschman (UCLA Medical School) independently reported the identification of an inducible mRNA for PGHS. This second isoform (PGHS-2) was shown to be inducible by mitogenic stimuli such as v-src, serum, forskolin, TPA, and endotoxin in a variety of cell lines. Moreover, elevated biosynthesis of prostaglandins in cells stimulated with mitogens correlated well with the increase in mRNA levels for the PGHS-2. Because of its potential role in the exaggerated biosynthesis of prostaglandins in response to inflammatory stimuli, PGHS-2 has quickly become the focus of attention for non-steroidal anti-inflammatory drug development. Lipoxygenases also metabolize polyunsaturated fatty acids to a host of physiologically active compounds such as leukotrienes, hydroxy fatty acids, etc. Soybean lipoxygenase is the first lipoxygenase to be characterized extensively both enzymologically and structurally. Unfortunately, most of the commercial preparations of this enzyme are mixtures of isoenzymes. We provide two of the highly purified isoenzymes of soybean lipoxygenase that are suitable for mechanistic studies. Attempts are underway to provide you with other biosynthetic enzymes of the leukotriene pathway.

Suggestions for further reading

Sloane, D.L., Leung, R., Craik, C.S., et al. A primary determinant for lipoxygenase positional specificity. Nature 354, 149-152 (1991).
Xie, W., Robertson, D.L., and Simmons, D.L. Mitogen-inducible prostaglandin G/H synthase: a new target for nonsteroidal anti-inflammatory drugs. Drug Development and Research 25, 249-265 (1992).

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