Amino Acid Residues in the Allosteric Site of<scp>l</scp>-Lactate Dehydrogenase from<i>Bifidobacterium longum</i>

Iwata, So; Minowa, Takashi; Sakai, Hiroshi; Ohta, Takao

doi:10.1080/00021369.1989.10869841

Agricultural and Biological Chemistry

1989

DOI: 10.1080/00021369.1989.10869841

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Amino Acid Residues in the Allosteric Site ofl-Lactate Dehydrogenase fromBifidobacterium longum

So Iwata

Takashi Minowa

Hiroshi Sakai

et al.

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1998

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“…The LDHs from some bacteria are allosteric enzymes exhibiting sigmoidal kinetics for pyruvate (homotropic activation), and are allosterically activated by fructose 1,6-bisphosphate (FBP) (heterotropic activation) (4), unlike non-allosteric vertebrate LDHs. Bifidobacterium longum L-lactate dehydrogenase is an FBP-dependent allosteric LDH (5,6). Abundant structural information on the enzyme has been obtained from its crystal structures (7,8).…”

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Homotropic Activation via the Subunit Interaction and Allosteric Symmetry Revealed on Analysis of Hybrid Enzymes ofl-Lactate Dehydrogenase

Fushinobu

Ohta²,

Matsuzawa

1998

Journal of Biological Chemistry

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L-Lactate dehydrogenase from Bifidobacterium longum shows homotropic activation by pyruvate as well as heterotropic activation by fructose 1,6-bisphosphate. Hybrid enzymes were produced from the wild-type subunit and a mutant subunit, whose substrate specificity was altered to that of malate dehydrogenase, and separated to analyze the substrate-induced homotropic activation mechanism. Oxamate, a competitive inhibitor of L-lactate dehydrogenase, was used to mimic the substrate-induced activation of the wild-type subunit as "a regulatory subunit." The malate dehydrogenase activity of the mutant subunit as "the catalytic subunit" of the hybrid enzymes was measured, and the activity of the mutant subunit was activated on the addition of oxamate. Thus, we directly observed the inter-subunit homotropic activation transmitted from the wild-type to the mutant subunit. Moreover, "isomeric" hybrid enzymes that have different structural subunit arrangements but identical subunit compositions showed identical kinetic natures. This indicates that the enzyme maintains its subunit symmetry during the allosteric transition.Most oligomeric allosteric proteins do not show hyperbolic Michaelis-type saturation curves, but sigmoidal ones in certain conditions. This phenomenon originates from the activation following an increase in the substrate concentration, and has been called "homotropic activation." Most kinetic studies on the homotropic activation of allosteric enzymes have focused on their sigmoidal substrate saturation curves. An alternative approach is analysis of the activation with the addition of low concentrations of "inhibitors" with subsaturating levels of substrates (1, 2). This activation indicates that the binding of an inhibitor to an active site affects the catalytic activity of neighboring active sites. With this method, however, complete analysis of the activation is quite complex, because the inhibitor and substrate compete at the active site.L-Lactate dehydrogenases (LDHs) 1 (EC 1.1.1.27) have been purified from a variety of organisms and tissues, and their structures and functions have been studied in detail (3). The active form of LDHs is a tetramer of identical 30 -35-kDa subunits. The LDHs from some bacteria are allosteric enzymes exhibiting sigmoidal kinetics for pyruvate (homotropic activation), and are allosterically activated by fructose 1,6-bisphosphate (FBP) (heterotropic activation) (4), unlike non-allosteric vertebrate LDHs. Bifidobacterium longum L-lactate dehydrogenase is an FBP-dependent allosteric LDH (5, 6). Abundant structural information on the enzyme has been obtained from its crystal structures (7,8). Four subunits of B. longum Llactate dehydrogenase are related by three molecular 2-fold axes named P, Q, and R, as for vertebrate LDHs (9). The tetramer has four active sites and two FBP-binding sites. The active site of each subunit lies near the Q-axis interface. On the other hand, the FBP-binding site is at the P-axis interface and is composed of residues from two subunits related by the P...

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Homotropic Activation via the Subunit Interaction and Allosteric Symmetry Revealed on Analysis of Hybrid Enzymes ofl-Lactate Dehydrogenase

Fushinobu

Ohta²,

Matsuzawa

1998

Journal of Biological Chemistry

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Amino Acid Residues in the Allosteric Site ofl-Lactate Dehydrogenase fromBifidobacterium longum

Cited by 1 publication

References 3 publications

Homotropic Activation via the Subunit Interaction and Allosteric Symmetry Revealed on Analysis of Hybrid Enzymes ofl-Lactate Dehydrogenase

Homotropic Activation via the Subunit Interaction and Allosteric Symmetry Revealed on Analysis of Hybrid Enzymes ofl-Lactate Dehydrogenase

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