Bridging in Vitro with in Vivo Enzymology

Abstract

Enzymes are extraordinary bio-catalysts that bridge the time-gap between otherwise slow chemical reactions and cellular processes. From an evolutionary perspective enzymes evolves with specific absolute values of their catalytic parameters kcat and KM. A fundamental question in biology is why specific absolute numbers of these parameters has evolved. In order to address this question for the enzyme adenylate kinase we have developed an in vivo enzymologi approach based on yeast genetics and NMR spectroscopy. In the approach yeast cell growth rates was analyzed in light of varying absolute values of kcat. A striking dependency of growth rates on kcat was discovered. On the other hand the dependency on KM is very weak. The data show that only around 10% of the kcat value is required for optimal cell growth under close to ideal growth conditions. The remaining 90% of the turn-over capacity may then be required for yeast to accommodate external stress conditions. In context of the weak dependency of growth rates on KM we have discovered a fundamental concept related to the specificity of the enzyme. Only the endogenous substrate (ATP) can fully activate the enzyme allosterically. Other chemically related substrates where binding is only marginally affected fail to fully activate the enzyme. The enzyme is thus catalytically selective while promiscuous with respect to binding. In summary, the approach developed herein has uncovered fundamental aspects of enzymatic catalysis inside living cells.