The effects of protein phosphorylation and acetylation on glycolytic rate-limiting enzymes and meat quality

Glycolysis is the primary way of energy metabolism in postmortem meat, and plays a critical role in the development of meat quality. Glycolysis is an enzymatically controlled process. Although hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) are regarded as the three glycolytic rate-limiting enzymes, the inner environment of postmortem muscle is different from that of living cells. Therefore, it is necessary to elucidate their roles in postmortem glycolysis. Identification of phosphorylated and acetylated sites on glycolytic enzymes in meat indicates that the activity of these enzymes may be regulated through protein post-translational modifications (PTMs). However, how protein phosphorylation and acetylation affect glycolytic enzymes in the postmortem muscle remains unclear. The aim of this study was to systematically explore the role of these three glycolytic rate-limiting enzymes and their PTMs in the postmortem muscle. First, we investigated the roles of HK, PFK and PK in glycolysis and meat quality by adding specific inhibitors to the postmortem muscle (Chapter 3). The results showed that PFK activity was significantly correlated with lactate content, but HK and PK activities showed a weak correlation with lactate content. PFK and PK exerted a greater impact on myofibrillar fragmentation index, protein degradation, and myoglobin status than HK, whereas HK exhibited a limited role in postmortem glycolysis and meat quality. Next, we ignored HK and investigated the effects of protein phosphorylation and acetylation on PFK and PK in vitro and in meat (Chapter 4). PFK and PK activities changed after the addition of phosphorylation and acetylation inhibitors to the meat homogenates. The correlation between glycolytic enzyme activity and PTMs was also displayed in postmortem meat with different glycolytic rates. Protein phosphorylation and acetylation had different cooperative patterns in the regulation of PFK and PK activities in postmortem meat. In addition, a possible PTM crosstalk was observed through protein acetylation increasing protein phosphorylation. Finally, we investigated the effects of specific PTMs sites on PFK and PK activities (Chapters 5 and 6). PFK_ T704, PFK_K678, PK_S99 and PK_K137 were tested. PFK_T704D decreased the catalytic efficiency by reducing the affinity between F6P. PK_K137Q inhibited glycolytic activity, possibly through a decrease in binding with PEP. PK_K137R might lessen its binding to actin, thus decreased PK-induced actin phosphorylation. In summary, this study broadens our insight into postmortem glycolysis and identifies important PTM sites for enzyme activity regulation, which provides a targeted strategy for meat quality preservation.