A new model for vitamin K-dependent carboxylation: the catalytic base that deprotonates vitamin K hydroquinone is not Cys but an activated amine.

Vitamin K-dependent (VKD) proteins require carboxylation for diverse functions that include hemostasis, apoptosis, and Ca(2+) homeostasis, yet the mechanism of carboxylation is not well understood. Combined biochemical and chemical studies have led to a long-standing model in which a carboxylase Cys catalytic base deprotonates vitamin K hydroquinone (KH(2)), leading to KH(2) oxygenation and Glu carboxylation. We previously identified human carboxylase Cys-99 and Cys-450 as catalytic base candidates: Both were modified by N-ethylmaleimide (NEM) and Ser-substituted mutants retained partial activity, suggesting that the catalytic base is activated for increased basicity. Mutants with Cys-99 or Cys-450 substituted by Ala, which cannot ionize to function as a catalytic base, were therefore analyzed. Both single and double mutants had activity, indicating that Cys-99 and Cys-450 do not deprotonate KH(2). [(14)C]NEM modification of C99A/C450A revealed one additional reactive group; however, Ser-substituted mutants of each of the eight remaining Cys retained substantial activity. To unequivocally test, then, whether any Cys or Cys combination acts as the catalytic base, a mutant with all 10 Cys substituted by Ala was generated. This mutant showed 7% wild-type activity that depended on factor IX coexpression, indicating a VKD protein effect on carboxylase maturation. NEM and diethyl pyrocarbonate inhibition suggested that the catalytic base is an activated His. These results change the paradigm for VKD protein carboxylation. The identity of the catalytic base is critical to understanding carboxylase mechanism and this work will therefore impact both reinterpretation of previous studies and future ones that define how this important enzyme functions.

Sir3p phosphorylation by the Slt2p pathway effects redistribution of silencing function and shortened lifespan.

An organism's lifespan is modulated by environmental conditions. When nutrients are abundant, the metabolism of many organisms shifts to growth or reproduction at the expense of longer lifespan, whereas a scarcity of nutrients reverses this shift. These correlations suggest that organisms respond to environmental changes by altering their metabolism to promote either reproduction and growth or long life. The only previously reported signaling mechanism involved in this response is the nutrient-responsive insulin/insulin-like growth factor-1 receptor pathway. Here we report another pathway that controls the length of yeast lifespan. Commitment to cell growth activates the Slt2p MAP kinase pathway, which phosphorylates the transcriptional silencing protein Sir3p, resulting in a shorter lifespan. Elimination of the Sir3p phosphorylation site at Ser275 extended lifespan by 38%. Lifespan extension occurs by a mechanism that is independent of suppressing rDNA recombination. Thus, Slt2p is an enzymatic regulator of silencing function that couples commitment to cell growth and shorter lifespan.