Lorne proteins: Among the enzymes

In the lead-up to the 2004 Lorne Protein, Cancer and Genome conferences, we continue our series of articles previewing some of the potential highlights at the events.

John Wallace, professor of biochemistry at the University's of Adelaide's Department of Molecular Bioscience, has been honoured for his achievements in enzymology with an invitation to deliver the 2004 Leach Lecture at Lorne.

Wallace studies biotin carboxylases. Four of these enzymes are constitutively expressed in human cells, and catalyse a variety of 'add-a-carbon-atom' reactions essential to cell metabolism, including lipogenesis, gluconeogenesis and the breakdown of amino acids.

Wallace says all are large, multi-subunit proteins with a number of highly conserved amino-acid sequence motifs, some of which occur in other enzyme families.

The limited high-resolution structural data available for the biotin-dependent enzymes indicates that they share highly conserved amino-acid sequence motifs with other enzyme families, including carbamoylphosphate synthetases and lipoyl-dependent enzymes, which catalyse similar, partial reactions.

Wallace says the challenge is to relate these structural features to the complex, multi-site reaction mechanisms that the enzymes catalyse, and their allosteric regulation.

Over the past eight years, Wallace has focused on biotin protein ligase (BPL), which catalyses the covalent attachment of biotin to enzymes. He says the enzyme catalyses the formation of a reactive intermediate between biotin and ATP, adenyl biotin.

Known to clinicians as holocarboxylase synthase, BPL in mutant form causes a relatively common inherited disorder in children, called multiple carboxylase deficiency. BPL mutations affect all four biotin-dependent enzymes, with the severity of the disorder depending on the particular mutation.

"The lucky ones can be treated with 10mg of biotin per day, to make the defective enzyme work, but some children are not so lucky, and their cells can't generate enough energy to function normally -- among other things, brain function can be severely affected," Wallace says.

He says a Canadian colleague, molecular geneticist Prof Roy Gravel, of the University of Calgary, has recently shown that BPL mutations can also disrupt biotinylation of histones, the supporting scaffolding for DNA in chromosomes.

Wallace says the BPL enzyme appears to play an important role in biotinylating histones, which may change the way they complex with DNA, affecting gene regulation and expression. This could explain why patients with severe BPL mutations show symptoms that are not easily explained by reductions in carboxylase reactions alone.

Proteomics hot spots

Protein hunters at Lorne can catch up with the latest technical tips and technological wrinkles at the 9th annual Lorne Proteomics Symposium at Erskine House from Friday, February 6 to Sunday February 8.

Organising committee member Dr Stuart Cordwell, of the Australian Proteome Analysis Facility, says there is likely to be interest in sessions on state-of-the-art technology, and proteomics without gels, where presenters will describe the latest techniques in 2D-PAGE gels and mass spectrometry.

Companies will present their latest developments in proteomics technology at the symposium's 'New on the Market' session.

Cordwell says proteomics is still hampered by the limited resolution of 2D gels -- cells typically express many more proteins that can be 'seen' on gels.

New techniques, like multi-dimensional chromatography and shotgun proteomics, can be used to analyse whole-cell extracts to detect elusive proteins whose properties causes them to 'fall off' 2D gels.

In shotgun proteomics, triptych digests of whole-cell extracts are run through two phases of high-pressure liquid chromatography, then analysed by mass spectrometer. The technique can identify proteins whose concentration, mass or charge make them undetectable by 2D gels.

The symposium will also devote a session on sub-proteomes -- the proteomics of organelles such as mitochondria and plastids, and membrane-bound and secreted proteins. There will also be sessions on clinical and pharmaceutical proteomics, biological applications of proteomics, and proteomics chips.