Affinity of Cystathionine β-Synthase for Pyridoxal 5′-Phosphate in Cultured Cells

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Abstract
Previous attempts to correlate in vivo pyridoxine-responsiveness with in vitro assays of cystathionine β-synthase activity in synthase-deficient homocystinuric patients have been only partially successful. All such studies, however, have been conducted with extracts of cultured skin fibroblasts grown in medium containing a high concentration (1,000 ng/ml) of pyridoxal. Having recently shown that such growth conditions may obscure important aspects of enzyme-coenzyme interactions by saturating most synthase molecules with their cofactor, pyridoxal 5′-phosphate, we have established conditions for growth of cells in pyridoxal-free medium. Under these conditions, intracellular pyridoxal 5′-phosphate fell by >95%, and saturation of cystathionine β-synthase apoenzyme with pyridoxal 5′-phosphate decreased from a predepletion value of 70% to Km of cystathionine β-synthase for pyridoxal 5′-phosphate in extracts of depleted cells from four in vivo-responsive patients was two to four times that of control. In contrast, the Km for pyridoxal 5′-phosphate in two lines from in vivo nonresponsive patients was 16- and 63-fold normal. These results suggest that cystathionine β-synthase activity in cells from patients containing a mutant enzyme with a moderately reduced affinity for pyridoxal 5′-phosphate can be increased by pyridoxine supplements in vivo, whereas that from patients whose enzyme has a more dramatically reduced affinity for the coenzyme cannot be so modulated because of limits on the capacity of such cells to accumulate and retain pyridoxal 5′-phosphate.