Activation of yeast hexokinase PII. Changes in conformation and association

Wilkinson, K.D.; Rose, I.A.

Journal of Biological Chemistry 254(6): 2125-2131

1979


ISSN/ISBN: 0021-9258
PMID: 33994
Accession: 068518590

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Abstract
Activation of yeast hexokinase [EC 2.7.1.1] PII at low pH by citrate or ATP was shown to occur at the monomer level, without requiring association to the dimer level. Thus, the previous suggestion that anonic activators of hexokinase, such as citrate and ATP, act by binding to the intersubunit site of the asymmetric dimer (BII) is unlikely. ATP also promotes formation of an inactivated dimer, and this inactivation can be reversed by citrate. The fact that ATP is not an activator at the high enzyme concentrations found in yeast suggests that the high rate of glycolysis is dependent on other positive effectors such as citrate. Sulfonated buffers such as Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) in the same pH range are shown to promote formation of an elongated dimer that may correspond to the BI crystal form described by Steitz et al. Upon activation by citrate, this dimer is partially dissociated to the active monomer. However, citrate must interact with the dimer and the monomer, because at high protein concentrations, saturating citrate cannot fully activate or dissociate the dimer stabilized by Hepes. Apparently the activation event is a conformational change that can occur at the monomer level and is stabilized by citrate or ATP. Sulfonated buffers inhibit by binding preferentially to the less active monomer conformation and prompting dimerization. At low pH and ATP concentration, the Hepes-stabilized dimer cannot exist in the activated form, although it may under other conditions. A model is presented to describe these interactions.