Putting the cart before the horse

Published on: Author: Colin Thorpe

When you know that a particular chemical reaction occurs in biology (e.g. glucose -> glucose-6-phosphate in glycolysis) you can use this activity to guide the isolation of the enzyme that catalyzes that transformation. During enzyme purification the aim is to winnow away the thousands of contaminating proteins leaving just the one you want. Traditionally, great emphasis has been placed on the need for purity – the late Efraim Racker opined “don’t waste clean thinking on dirty enzymes”!

While most enzymes known today were initially purified with prior knowledge of their likely substrates and products, and with a good idea of their place in metabolism, a few were isolated first as proteins. Knowledge of their enzymatic activity came later – putting the cart before the horse as it were. Our discovery of the QSOX family of enzymes came about in this back-handed way.

A colleague, Hal White, told us of his finding that egg white contains FAD – a derivative of the vitamin riboflavin that is usually associated with redox enzymes.

We jointly wondered “why is FAD in egg white” since no FAD-dependent enzyme was known from this source. Detractors of this type of non-hypothesis-driven research want an overarching hypothesis and an encirclement of specific aims to provide intellectual cover. We had none of this – but answering the “why is FAD in egg white” question has led to the discovery of two new enzyme families, to an intriguing new disulfide-rich biomaterial and to suggestions of a new strategy to slow the growth of human tumors. At the end of this blog post there are links to provide an entrée to some of these aspects.

So, out of curiosity, we purified the protein binding FAD in egg white. We did not know whether the protein would prove to be an enzyme – so we had no enzyme activity to follow. But FAD is yellow and that’s what we followed.   In crude egg white only about one protein molecule out of about 10,000 was the FAD-binding protein we sought – but we were able fish out a small amount of a pure bright yellow enzyme from a large volume of chicken egg white.

So now we had the protein pure – what to do next? Since flavins are usually involved in redox reactions we tested a number of likely reducing substrates. We screened candidate substrates by adding them to the yellow protein one by one. We were looking for a color change because a real substrate of the enzyme would likely modify the visible spectrum of the enzyme – subtle changes would require a spectrophotometer to detect – major changes would be evident by eye. After trying a number of potential substrates that had no effect on the spectrum, we found that thiol-containing compounds induced dramatic changes in color of the flavin cofactor bound to the protein. In the figure you can see stills taken from a video showing the bright yellow color of the enzyme flash through a blue tint on its way to paler yellow as oxygen is depleted from the solution.

So this was the essential clue that led us to the discovery of a new family of sulfhydryl oxidase enzymes. The chemistry was typical of sulfhydryl oxidases that use that yellow FAD cofactor to catalyze the oxidation of thiols in the presence of molecular oxygen.

2 R-SH   +    O2        ->     R-S-S-R      +      H2O2

We then surveyed the range of thiol-containing substrates that the enzyme could process in pure solution. We found that the enzyme could oxidize a wide range of –SH groups found in both small and large molecules. Every protein we tested was a substrate of the enzyme providing that it was flexible enough to present two or more cysteine side chains in a flexible environment – so reduced unfolded proteins (prepared by breaking their native disulfide bonds prior to assay) were excellent substrates of the enzyme. The enzyme worked at rates that were far greater than other candidates for disulfide bond generation in higher eukaryotes.

Fortunately the catalyst we had stumbled upon in egg white is not just for the birds! QSOX enzymes (the official abbreviation of the Quiescin-sulfhydryl oxidases) are found in almost all non-fungal eukaryotes – from small unicellular algae to humans. So what are these ancient enzymes doing? It is probably safe to say that the role of QSOX enzymes is to make disulfides (with the possible ancillary function of generating hydrogen peroxide) – but what disulfides exactly, and in what cellular or tissue locales?

So we have done things backwards – we found an enzyme by accident and then constructed a story of a likely physiological role based on its prodigious ability to make disulfide bonds in protein substrates in the laboratory. Now researchers are searching for the molecular roles of QSOX in cells and tissues. For example QSOX enzymes have been shown by the Bulleid laboratory to be very efficiently secreted and the work of the Fass and Lake groups have provided intriguing evidence for a major extracellular role of QSOX in the extracellular matrix. Several studies now associate the up-regulation of QSOX with an adverse outcome in certain cancers.

Why, we wonder, is QSOX secreted into blood, sweat and tears! – is it an incidental product of the secretory machinery, or is QSOX doing something useful in biological fluids? Finally what is the role of QSOX in unicellular organisms – from benign marine algae to the pathogenic trypanosoma?

Lots of important questions – and we still don’t know why QSOX is secreted into egg white!

Of possible interest …

Discovery of QSOX – the real story ([caution: gripe] to say that QSOX was “isolated more than three decades ago” does not, in the writer’s opinion, do justice to the facts of the case!).  For a history of the early developments see QSOX-ology.

QSOX and cancer

CREMP: an amazing disulfide-rich biomaterial

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