Question

8. Practice 6: Determination of LDH activity A. Pre-lab questions: 1) Which substrate should be used when NAD' is added to the LDH reaction? Explain 2) Why the spectrophotometer is fixed at 340 nm? Explain

Answer 1

1)L-lactate dehydrogenase (L-LDH) catalyzes the interconversion of pyruvate and NADH+ to L-lactate and NAD+.
H-lactate dehydrogenase (H-LDH) catalyzes the interconversion of D-lactate and ferricytochrome c to pyruvate and ferrocytochrome c.
Lactate Dehydrogenase (LDH) is an important enzyme in humans. It occurs in different regions of the body, each region having a unique conformation of different subunits. LDH is a key enzyme in anaerobic respiration. Anaerobic Respiration is the conversion of pyruvate into lactate acid in the absence oxygen. This pathway is important to glycolysis in two main ways. The first is that if pyruvate were to build up glycoysis and thus the generation of ATP would slow. The second is anaerobic respiration allows for the regeneration of NAD+ from NADH. NAD+ is required when glyceraldehyde-3-phosphate dehydrogenase oxidizes glyceraldehyde-3-phosphate in glycolysis, which generates NADH. Lactate dehydrogenase is responsible for the anaerobic conversion of NADH to NAD+.

L-lactate H OHH Pyruvate CHs COO L-LDH D-LDH CH3 + NADH + H+ NAD+ + Keq = 10+13 M-1 COO D-lactate HO. H CH3 COO

Title: Glycolysis and Gluconeogenesis Availability: CC BY 2.0 Last modified: 2/21/2013 Organism: Homo sapiens SLC2A1 SLC2A2 SLC2A3 SLC2A4 SLC2A5 Glycolysis Gluconeogenesis HK1 G6PC HK3 Glucose-6P Pentose Phosphate Pathway Glycogen metabolism Fructose 6P PFKM PFKL PFKP FBP1 FBP2 Fructose-1,6BP ALDOA ALDOB ALDOC Glyceraldehyde 3FP Dihydroxyacetone-P Triglyceride synthesis GAPDHS GAPDH 1,3BP-Glycerate PGK1 PGK2 Cytosol 3P-Glycerate Mitochondrion PGAM2 PGAM1 2P-Glycerate ENO1 ENO3 ENO2 Aspartate Aspartate P-enolpyruvate GOT1 GOT2 PKM2 PKM2 PKLR Oxaloacetate Oxaloacetate РСК 1 MDH1 PC MDH2 Malate Malate Pyruvate LDHAL6B LDHA LDHB LDHC MPC1 Pyruvate MPC2 Lactate PDHA1 PDHA2 PDHB DLAT DLD PDHX TCA Cycle Acetyl-CoA

The enzyme has a catalytically-active histidine residue and requires NAD*/NADH as a coenzyme. он NAD+/NADH,H+ 0- 0- lactate dehydrogenase lactate O pyruvate O

An enzyme balances two apparently conflicting requirements to function properly. In forming the so-called Michaelis complex, the bound substrate is positioned within the protein in close contact with key protein groups that facilitate catalysis. Additionally for bimolecular reactions, the two bound substrates are held tightly together and positioned correctly for chemical reaction. Generally, static pictures involving no motion of the reacting groups are used to yield working mechanistic pictures. On the other hand, a second requirement of enzymatic catalysis is effective substrate binding and, the reverse, product release. Substrate is captured from solution and shuttled in and out of the binding pocket of the active site in a timely manner, typically on the order of a millisecond. Binding is necessarily a dynamical process. Substantial motions within the protein complex are required, including the recruitment of key proteins groups into the active site and desolvation and closure of the binding pocket. This often involves the motion of an active site loop, wherein an open form can facilitate ligand binding and release, and a closed form prepares, controls, and protects the reacting species. The dynamics of ligand binding to proteins is little understood, but involve motions from femtoseconds to tens of milliseconds (and sometimes even longer), and the process for enzymes is such that the enzyme·substrate complex lives just long enough to permit effective catalysis and no longer. The goal of this work is to examine the dynamics of how the Michaelis complex is formed in lactate dehydrogenase (LDH). We focus on how the enzyme·ligand encounter complex is formed; the encounter-complex species is of special importance in understanding the binding process.

LDH catalyzes the direct transfer of a hydride ion from the pro-R face of the reduced nicotinamide group of NADH to the C₂ carbon of pyruvate producing NAD+ and the alcohol lactate, accelerating the solution chemical reaction by some 14 orders of magnitude . Binding of substrate to LDH is ordered and follows the formation of the LDH/NADH binary complex. The substrate binding pocket lies deep within the protein, buried ∼10 Å from the protein's surface , although our recent molecular dynamic calculations suggest that the protein samples conformations wherein the binding pocket is substantially exposed to solvent . It supplies the catalytically crucial His, and the preformed pocket additionally solvates the substrate's charged carboxyl group by supplying Arg. The rate-limiting step in the turnover of LDH is not the chemical hydride transfer step but rather loop motion involving closure of the so-called mobile loop (surface residues 98–110), occurring in a time of ∼1–10 ms

2) Many biological assays have as their basis a link to the oxidative status of nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP). Many dehydrogenase enzymes use these coenzymes to transfer hydrogen groups between molecules. Because the reduced forms of these molecules differ from the oxidized forms in their ability to absorb light, it is possible to quantitate reactions based on light absorbance at 340 nm or by the fluorescent emission of light at 445 nm. Here we describe the use of the Synerg 2 Multi-Detection Microplate Reader to quantitate NADH using either fluorescence or absorbance modes. Introduction Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are soluble dinucleotides that can be reversibly reduced by the addition of 2 hydrogen ions. While both molecules act as coenzymes in reversible reactions, they are involved in different types of reactions. NAD is generally used as an acceptor of reducing equivalents in catabolism, particularly glycolysis, the tricarboxylic acid cycle, and β-oxidation of fatty acids, while NADH, is reoxidized by complex I of the electron transport chain or by dehydrogenase enzymes during anaerobic metabolism (1). NADP is characteristically involved with reductive synthesis reactions, such as fatty acid and steroid synthesis (1). As demonstrated in Figure 1, NAD is a multiple ringed structure, which undergoes redox reactions within its nicotinamide ring (2). The closely related NADP molecule is phosphorylated on the 2’ position of the adenosine ribose ring. (2). In terms of quantitation, enzymatic dehydrogenase reactions involving NAD or NADP take advantage of the property of the reduced forms, NADH or NADPH, to absorb light at a wavelength of 340 nm while the oxidized forms do not. Likewise, the reduced forms are capable of fluorescent emission at 445 nm when excited at 340 nm, while the oxidized forms are not (4)

LDH pyruvate L-lactate NH2 NADH NAD

Lactate dehydrogenase is the most important clinically of several dehydrogenases occurring in human serum. Lactate dehydrogenase is cytoplasmic in its cellular location and in any one tissue is composed of one or two of five possible isoenzymes. While many of its clinical applications involve quantification of one or more specific serum isoenzymes, an estimate of total LD is required usually. Lactate dehydrogenase catalyzes the reversible reaction: L-lactate + NAD+ in equilibrium pyruvate + NADH. The bidirectional reaction is monitored spectrophotometrically by measuring either the increase in NADH at 340 nm produced in the lactate-to-pyruvate reaction (L----P) or by the decrease in NADH at 340 nm produced in the pyruvate-to-lactate (P----L) reaction. Kinetic assay systems for the measurement of the reaction system in both directions are comprehensively reviewed as well as the standardization efforts proposed to date.