Differences and similarities in binding of pyruvate and l-lactate in the active site of M4 and H4 isoforms of human lactate dehydrogenase

Abstract
We present QM/MM calculations that show differences in geometries of active sites of M4 and H4 isoforms of human LDH ligated with oxamate, pyruvate or l-lactate. As the consequence of these differences, binding isotope effects of the methyl hydrogen atoms of pyruvate and l-lactate may be used to experimentally distinguish these isoforms. Based on the FEP calculations we argue that l-lactate is a better candidate for the experimental studies. Our calculations of energies of interactions of ligands with the active site residues provide explanation for the observed experimentally sensitivity to inhibition of the M4 isoenzyme isoform and pinpoint the differences to interactions of the ligand with the histidine residue. We conclude that pyruvate interacts much stronger in the active site of H4 than M4 isoform and that the latter interactions are weaker than with water molecules in the aqueous solution

Research highlights
► Isoforms M4 and H4 of human lactate dehydrogenase exhibit different BIEs of pyruvate and l-lactate. ► Hydrogen BIEs of l-lactate and pyruvate can be used for differentiation of the LDH isoforms. ► Affinities of pyruvate toward active sites of M4 and H4 are different. ► l-lactate is a good candidate for experimental differentiation of LDH isoforms.

Conclusions
Studies presented herein explain the experimental observations where H4 isoform is much more sensitive to inhibition than the M4 isoenzyme. The studies of energies of interactions of ligands with the active site residues provide explanation to this problem and pinpoint the differences to interactions of pyruvate with the histidine residue. Our theoretical studies show that pyruvate interacts much stronger in the active site of H4 than M4 isoform. Moreover, the interactions of pyruvate with active site of M4 isoform are weaker that with water molecules in the aqueous solution.

Using QM/MM calculations it was shown that the geometries of the LDH active sites are significantly different when oxamate, pyruvate or l-lactate are ligated. Because of these differences M4 and H4 isoforms of human LDH may be distinguished on the basis of binding isotope effects of hydrogen atoms of the methyl group of pyruvate and l-lactate. FEP calculations indicate that pyruvate has low affinity to the active site of the M4 isoform thus it seems safer to use l-lactate as a ligand for experimental differentiation the LDH isoforms.



Archives of Biochemistry and Biophysics
Volume 505, Issue 1, 1 January 2011, Pages 33-41
Special Section: Trends in Enzymology 2010

Increased myeloperoxidase enzyme activity in plasma is an indicator of inflammation and onset of sepsis

Circulating lipopolysaccharides released from bacteria may activate both neutrophils and monocytes. The activated neutrophils release myeloperoxidase (MPO), a specific enzyme with strong oxidative activity. The aim of this study was to evaluate MPO enzyme activity in plasma of critically ill patients and to check the hypothesis that these concentrations in plasma would be higher in sepsis and systemic inflammatory conditions, as neutrophils release their contents before proliferating in response to stress.

Results
The plasma MPO enzyme activity in sepsis patients was significantly higher than that in the control group (mean, 2.4 ± 1.8 in sepsis and 1.86 ± 1.2 nmol per milligram protein per 10 minutes in systemic inflammatory response syndrome vs 0.32 ± 0.11 nmol per milligram protein per 10 minutes in healthy controls). Mean plasma lactate levels in sepsis (7.8 ± 1.2 mmol/L) and shock patients (9.5 ± 1.2 mmol/L) and cytokines like tumor necrosis factor–α, interleukin-8, and interleukin-1β were simultaneously evaluated to establish onset of inflammation and sepsis. These results show that neutrophil activation occurring during inflammation and sepsis could be detected by plasma MPO concentration

Conclusion : The plasma MPO concentrations may be a marker of the neutrophil proliferation and severity of inflammation

a Department of Anaesthesia, Chatrapati Shahuji Maharaj Medical University, Lucknow, UP, India

b Pharmacology Division, Central Drug Research Institute, Lucknow, UP, India