Properties of Normal and Mutant Recombinant Human Ketohexokinases and Implications for the Pathogenesis of Essential Fructosuria

Abstract

Alternative splicing of the ketohexokinase (fructokinase) gene generates a "central" predominantly hepatic isoform (ketohexokinase-C) and a more widely distributed ketohexokinase-A. Only the abundant hepatic isoform is known to possess activity, and no function is defined for the lower levels of ketohexokinase-A in peripheral tissues. Hepatic ketohexokinase deficiency causes the benign disorder essential fructosuria. The molecular basis of this has been defined in one family (compound heterozygosity for mutations Gly40Arg and Ala43Thr). Here we show that both ketohexokinase isoforms are indeed active. Ketohexokinase-A has much poorer substrate affinity than ketohexokinase-C for fructose but is considerably more thermostable. The Gly40Arg mutation seems null, rendering both ketohexokinase-A and ketohexokinase-C inactive and largely insoluble. The Ala43Thr mutant retains activity, but this mutation decreases the thermal stability of both ketohexokinase-A and ketohexokinase-C. At physiologic temperature, this results in significant loss of ketohexokinase-C activity but not of ketohexokinase-A. Affected individuals who carry both mutations therefore probably have a selective deficiency of hepatic ketohexokinase, with peripheral ketohexokinase-A being preserved. These findings raise the possibility that ketohexokinase-A serves an unknown physiologic function that remains intact in essential fructosuria. Further mutation analysis in this rare disorder could illuminate the question of whether ketohexokinase-A activity is, unlike that of ketohexokinase-C, physiologically indispensable.

Opposing effects of fructokinase C and A isoforms on fructose-induced metabolic syndrome in mice

“…KHK-C has been reported to have much greater affinity for fructose than KHK-A, raising the question of whether KHK-A actively metabolizes fructose in vivo (22). We confirmed the lower K m of KHK-C compared with KHK-A using human recombinant proteins (Fig.…”

“…KHK-C is expressed primarily in the liver, kidney, and intestines, whereas KHK-A is more ubiquitous (21). Whereas both KHK-C and KHK-A can metabolize fructose, KHK-C is considered the primary enzyme involved in fructose metabolism due to its lower K m (22). Because of its higher K m , KHK-A is not considered presently to be actively involved in fructose metabolism but rather has been postulated to act on as yet unknown substrates (22).…”

Divergent effects of glucose and fructose on hepatic lipogenesis and insulin signaling

“…KHK catalyzes the first step of intracellular fructose metabolism (26). Khk expression was increased 2-fold in mice supplemented with fructose on a chow diet and 3-fold in mice supplemented with fructose on an HFD, as assessed by quantitative PCR (qPCR) ( Figure 5A) or RNA-seq ( Figure 4C).…”

Fructose consumption as a risk factor for non-alcoholic fatty liver disease

“…To measure enzymatic activity of KHK, we used a coupled enzymatic assay based on existing methods [35,36,37]. Cells were harvested on ice in the buffer containing 25 mM HEPES (pH 7.1), 100 mM KCl, 1 mM DTT, 0.1 mM EDTA and homogenized by 100 strokes of the pestle.…”