Structure‐Function Relationship in the Allosteric <scp>l</scp>‐Lactate Dehydrogenases from <i>Lactobacillus casei</i> and <i>Lactobacillus curvatus</i>

The activation of the allosteric l-lactate dehydrogenases from Lactobacillus casei and LactOBacillus curvatus induced by binding of the effectors fructose 1,6-bisphosphate [Fru(1,6)P2] and Mn2+ is correlated with conformational changes as indicated by alterations of the tryptophan and tyrosine absorption and by an alteration of the triespophan fluorescence of the proteins.

Abstract

The activation of the allosteric l ‐lactate dehydrogenases from Lactobacillus casei and Lactobacillus curvatus induced by binding of the effectors fructose 1,6‐bisphosphate [Fru(1,6) P 2 ] and Mn 2+ is correlated with conformational changes as indicated by alterations of the tryptophan and tyrosine absorption and by an alteration of the tryptophan fluorescence of the proteins. Both enzymes contain four NADH and Fru(1,6) P 2 binding sites per tetramer as determined by fluorescence measurements. In the case of the L. casei enzyme at least four Mn 2+ binding sites were determined for the tetrameric state by electron spin resonance (ESR) spectroscopy. The modification of tryptophan of the L. casei enzyme with dimethyl‐(2‐hydroxy‐5‐nitrobenzyl)‐sulfonium bromide suggests that the alteration of the tryptophan absorption is due to a tryptophan residue being located in the interior of the protein, whereas the alteration of the tryptophan fluorescence is due to a second tryptophan residue which is located on the surface of the enzyme. Thus the effector‐induced conformational changes may cause structural alterations in an inner as well as in an outer region. To obtain information about the distance between the coenzyme and Mn 2+ binding sites, ESR spectra were recorded of the spin‐labeled NADH analogs bound to the L. casei enzyme in the presence and absence of Mn 2+ . The analogs were substituted at C‐6 or C‐8 with a 4‐(2,2,6,6‐tetramethyl‐piperidinyl‐1‐oxyl)‐amino group. The oxidized forms of both derivatives, labeled by a nitroxide radical, were shown to be active coenzymes. However, no spin‐spin interaction between the spin label and Mn 2+ could be observed, indicating that the Mn 2+ binding site is at least 1.5–2.0 nm apart from the adenine moiety of the coenzyme. Although a direct interaction between the metal and coenzyme is unlikely because of this large distance, binding of the effectors to the L. casei enzyme causes changes of the fluorescence of enzyme‐bound NADH. Thus, the bound coenzyme appears to be affected by the conformational changes in the L. casei l ‐lactate dehydrogenase induced by Fru(1,6) P 2 and Mn 2+ .