Studies on the Active Site of Yeast Hexokinase. Specific Phosphorylation of a Serine Residue Induced by d-Xylose and ATPMg

Abstract

Yeast hexokinase A (ATP : D-hexose 6-phosphotransferase) is inactivated when incubated in the presence of xylose and ATPMg, or in the presence of D-lyxose in a reaction medium in which ATPMg is being continuously regenerated (phosphoenolpyruvate and pyruvate kinase).The inactivation is due to the phosphorylation of the protein. A linear relationship was observed between the inactivation and the incorporation of 32P from [p3'P]ATP. All hexokinase and ATPase activity of the enzyme is lost when one phosphoryl group is incorporated per enzyme subunit (molecular weight 51 000). The phosphoryl group is covalently bound by a ester linkage with a serine residue of the protein.DelaFuente et al. [2] have shown that D-xylose or D-lyxose, which are non-phosphorylatable inhibitors of glucose phosphorylation by yeast hexokinase, can enhance the ATPase activity of the enzyme. Thus 0.1 M lyxose cause an approximately 20-fold increase in V and a 40-fold decrease in K, for ATP hydrolysis. Furthermore DelaFuente [ 11 observed that D-xylose has only a transitory activation effect which is then followed by inactivation of the enzyme. These authors suggested that the binding of the two pentoses at the enzyme active site involves an 'induced-fit' which changes the protein conformation. In a preliminary communication, Cheng et al. [3] indicated that the inactivation induced by D-xylose was due to the phosphorylation of the protein.It is thus interesting to determine which amino acid residue of yeast hexokinase is phosphorylated in the course of the specific inactivation process induced by xylose and ATPMg. In this paper we report a kinetic study relating the loss of the enzyme activity and the phosphorylation of hexokinase induced by xylose and ATPMg. In addition we present experimental evidence indicating that the phosphoryl group is linked to the protein by an ester bond with the primary hydroxyl 'group of a serine residue. Furthermore we observed that lyxose can also induce the inactivation of yeast hexokinase, in the presence of ATPMg.A preliminary report of a portion of this work has already been given [4]. EXPERIMENTAL PROCEDURE MaterialsBaker's yeast hexokinase A was purified as described by Rustum et al. [5] with some modifications. The last two steps on DEAE-cellulose were replaced by a fractionation by isoelectric focusing, using a pH gradient of 0.5 pH unit (pH 4.75 to pH 5.25). Our enzyme preparation had a specific activity of 150 units/mg at 25 "C and was homogeneous both by disc electrophoresis and by ultracentrifugation.Pyruvate kinase and phosphoenolpyruvate dicyclohexylammonium salt were obtained from Boehringer Mannheim. The nucleotides were purchased from P.L. Biochemicals Co. [ Y -~~P ] A T P was purchased from the Commissariat a 1'Energie Atomique. Lyxose was obtained from Fluka; xylose and fructose from Merck; glucose and mannose from Prolabo ; charcoalimpregnated filter paper from Labo-Moderne (Paris) ; disc filter paper (2.4 cm diameter) 3MM from Whatman, 0-phospho-DL-serine and DL-0-phosphothreonine were purch...

Inhibition and Inactivation of Glucose-phosphorylating Enzymes from Saccharomyces cerevisiae by D-Xylose

“…2a) are in agreement with those previously described for the animal enzyme (Lazo & Sols, 1979). The inactivation of the enzyme depended on the presence of ATP, suggesting that the mechanism of inactivation could be a phosphorylation of the protein (Menezes & Pudles, 1976). On the other hand, hexokinase PI1 inactivation by D-xylose was independent of ATP ( Fig.…”

“…A decrease in hexokinase PI1 activity induced by D-xylose leads to the relief of carbon catabolite repression ot'invertase ( F e r n h d e z et ul., 1984). On the other hand, a highly specific inactivation of yeast and animal hexokinase induced by D-XylOSe has been reported (De la Fuente et al, 1970;Lazo & Sols, 1979) which is said to involve a potentially reversible paracatalytic phosphorylation of the enzyme a t a serine residue (Menezes & Pudles, 1976). Xylose is a non-phosphorylatable glucose analogue that competitively inhibits the hexokinase reaction (De la Fuente et d .…”

Phosphorylation of yeast hexokinases

“…Catalysis by yeast hexokinase does not involve a phosphoenzyme intermediate [22]. D-Xylose causes inactivation of hexokinase in vivo [23], and D-xylose-stimulated inactivation and phosphorylation in vitvo are known for both hexokinase 1 [24] and hexokinase 2 [25]. Protein kinase activity has also been reported for the latter enzyme [26].…”

The residual enzymatic phosphorylation activity of hexokinase II mutants is correlated with glucose repression in Saccharomyces cerevisiae.

“…For example, hexokinase II might be involved in the generation of a phosphorylated metabolite. Hexokinase is known not to undergo autophosphorylation in normal catalysis but will inactivate itself by autophosphorylation in the presence of certain pentoses, such as xylose (5,11,23).…”