A generic HTS assay for kinase screening: Validation for the isolation of an engineered malate kinase

Abstract

An end-point ADP/NAD(+) acid/alkali assay procedure, directly applicable to library screening of any type of ATP-utilising/ADP producing enzyme activity, was implemented. Typically, ADP production is coupled to NAD(+) co-enzyme formation by the conventional addition of pyruvate kinase and lactate dehydrogenase. Transformation of enzymatically generated NAD(+) into a photometrically active alkali derivative product is then achieved through the successive application of acidic/alkali treatment steps. The assay was successfully miniaturized to search for malate kinase activity in a structurally-guided library of LysC aspartate kinase variants comprising 6,700 clones. The screening procedure enabled the isolation of nine positive variants showing novel kinase activity on (L)-malate, the best mutant, LysC V115A: E119S:E434V exhibited strong substrate selectivity for (L)-malate compared to (L)-aspartate with a (k(cat)/K-m)(malate)/(k(cat)/K-m)(aspartate) ratio of 86. Double mutants V115A:E119S, V115A:E119C and E119S:E434V were constructed to further probe the origins of stabilising substrate binding energy gains for (L)-malate due to mutation. The introduction of less sterically hindering side-chains in engineered enzymes carrying E119S and V115A mutations increases the effective volume available for substrate binding in the catalytic pocket. Improved binding of the (L)-malate substrate may be assisted by less hindered movement of the Phe184 aromatic side-chain. Additional favourable long-range electostatic effects on binding arising from the E434V surface mutation are conditionally dependent upon the presence of the V115A mutation close to Phe184 in the active-site