Influence of pH on the manganese(2+) activation of and binding to yeast enolase: a functional study

Abstract

The influence of pH on the activation of yeast enolase by Mn2+ was measured by steady-state kinetics. The pH influence on the binding of Mn2+ to apoenolase and the enolase-substrate complex was measured by EPR spectroscopy. At pH values above 6.6, activation by Mn2+ is fit by Michaelis-Menten kinetics, but at higher concentrations of Mn2+, inhibition is observed. Under conditions analogous to the kinetic studies, the enzyme binds two Mn2+ per dimer with a Kd in the micromolar range. In the presence of the substrate 2-phosphoglycerate, three thermodynamically distinct cation binding sites per monomer are detected and the binding constants are determined by a fit to the data. As the pH decreases, the reaction velocity decreases and the cation inhibition becomes minimal. Under these conditions, only two Mn2+ binding sites per monomer are observed; the third site must be the inhibitory site. The velocity and kinetic constants are minimally affected by buffer except at pH 5.8 with PIPES. Under these conditions, the velocity is only about 40% that observed with other buffers and only a single binding site for Mn2+ per monomer is detected in the presence or absence of substrate. A direct role in the catalytic mechanism by the second cation is called to question. The binding constant for Mn2+ at site I is independent of pH over the range from 7.5 to 5.2, and the binding at site II increases only slightly over this same pH range. These results indicate that the cation sites at positions I and II contain ligands that are pH independent over this range.(ABSTRACT TRUNCATED AT 250 WORDS)

Standardized Assay Medium To Measure Lactococcus lactis Enzyme Activities while Mimicking Intracellular Conditions

“…Activities of other enzymes did not differ greatly in the presence of small amounts (1 to 5 mM) of ammonium sulfate. For ENO, an alternative assay (26) measuring PEP absorbance at 240 nm is a more elegant way to solve the ammonium sulfate problem if measurements in the UV range are possible. In conclusion, ammonium sulfate could have an effect on enzyme activities, and it is better to work with desalted enzymes.…”

Cloning, expression and characterization of an extracellular enolase fromLeuconostoc mesenteroides

“…For most enolases the most effective metal activator is Mg 2+ (Wang & Himoe, 1974; Pietkiewicz & Kustrzeba‐Wójcicka, 1983; Lee & Nowak, 1992a, b). Mg 2+ strongly activates enolase from L .…”

“…mesenteroides B‐512FMCM, but Mn 2+ and Zn 2+ are at least equally effective. Mn 2+ strongly activates Candida albicans and Saccharomyces cerevisiae enolases (Lee & Nowak, 1992a, b), but is a weaker activator of the enolase from carp muscle. Zn 2+ induces higher activity than Mn 2+ in carp enolase, but the converse is true for the enolases of yeast and C. albicans (Pietkiewicz & Kustrzeba‐Wójcicka, 1983; Lee & Nowak, 1992a, b).…”

“…Mn 2+ strongly activates Candida albicans and Saccharomyces cerevisiae enolases (Lee & Nowak, 1992a, b), but is a weaker activator of the enolase from carp muscle. Zn 2+ induces higher activity than Mn 2+ in carp enolase, but the converse is true for the enolases of yeast and C. albicans (Pietkiewicz & Kustrzeba‐Wójcicka, 1983; Lee & Nowak, 1992a, b). In much of the literature concerning enolases, the metal ion that binds with highest affinity is called the ‘conformational’ or ‘structural’ metal ion, whereas the metal ion that binds with lower affinity is the ‘catalytic’ metal ion (Faller et al , 1977; Brewer, 1985).…”

Crystallization at low salt concentration and alkaline pH and preliminary crystallographic data for a monoclinic form of yeast enolase

“…Studies of Cd 2+ and Mn 2~ complexes of yeast enolase using ~I3Cd2*NMR and EPR spectroscopy (Spencer, Brewer & Ellis, 1985;Lee & Nowak, 1992a) indicated that the ligands at the 'catalytic' site are all O atoms and that the 'catalytic' metal ion appears to be more than 12 from the 'conformational' site (Lee & Nowak, 1992b). In contrast, studies of the bis Mn 2* complex of phosphonoacetohydroxamate in the active site of enolase suggested that the two metal ions are bridged by a single O atom of this very strongly bound inhibitor (Poyner & Reed, 1992).…”