Epistatic interactions between mutations are thought to play a crucial role in a number of evolutionary processes, including adaptation and sex. Evidence for epistasis is abundant, but tests of general theoretical models that can predict epistasis are lacking. In this study, I test the ability of metabolic control theory to predict epistasis using a novel experimental approach that combines phenotypic and genetic perturbations of enzymes involved in gene expression and protein synthesis in the bacterium Pseudomonas aeruginosa. These experiments provide experimental support for two key predictions of metabolic control theory: (i) epistasis between genes involved in the same pathway is antagonistic; (ii) epistasis becomes increasingly antagonistic as mutational severity increases. Metabolic control theory is a general theory that applies to any set of genes that are involved in the same linear processing chain, not just metabolic pathways, and I argue that this theory is likely to have important implications for predicting epistasis between functionally coupled genes, such as those involved in antibiotic resistance. Finally, this study highlights the fact that phenotypic manipulations of gene activity provide a powerful method for studying epistasis that complements existing genetic methods.