Research Fellow The University of Western Australia
In all cellular organisms, enzyme pools are continually renewed through a cyclic process of synthesis, degradation and re-synthesis (enzyme turnover). However, the factors and mechanisms that control this process are poorly understood. It is unclear why some enzymes turn over faster than others and how ‘old’ enzymes differ from ‘new’ enzymes structurally and functionally. To unlock the mysteries of this process, we considered enzyme turnover rates (measured by stable isotope labelling) and metabolic flux rates (calculated using flux balance analysis) in plants. Considering these rates together as a unitless ratio of catalytic-cycles-till-replacement (CCR) provides a new quantitative tool to assess the replacement schedule of and energy investment into enzymes as they relate to function. We assessed CCRs of selected enzymes in plants (and bacteria for comparison) to reveal a range of seven orders of magnitude for this ratio. These values will allow genetic engineers and synthetic biologists to seek CCR-based improvements in crop productivity. To better understand the structural and functional differences between newly-synthesised enzymes and enzymes that have existed in cells for some time, we investigated tagging and enriching nascent enzymes using azidohomoalanine (AHA) and homopropargylglycine (HPG). These methionine surrogates have previously been used for enzyme tagging in mammalian systems and we assessed their suitability for this purpose in plants (proteins tagged with AHA or HPG can be separated from the rest of the protein pool using commercially-available kits). Unlike animal cells, plant cells synthesise methionine, which potentially complicates the use of such surrogates in plants. We show that AHA and HPG influence the cellular levels of methionine and related metabolites in Arabidopsis. We also show the effects of AHA and HPG on cell growth, the enzymes in plants that can be shown to incorporate AHA and HPG and discuss the efficiency of this approach in plants vs. mammals.
Coauthors: Andrew Hanson – University of Florida;Christopher Henry – Argonne National Laboratory;Adrian Hegeman – University of Minnesota;Harvey Millar – The University of Western Australia