DETERMINATION OF GLUTAMATE DEHYDROGENASE ACTIVITY Glutamate dehydrogenase (GDH) (E.C. 1.4.1.3) is a hexameric enzyme that catalyzes the reversible conversion of L-glutamate to α-ketoglutarate and ammonia while reducing NAD(P)+ to NAD(P)H/H+ as coenzymes (Figure 1.). It is found in all living organisms serving both catabolic and anabolic reactions. In mammalian tissues, oxidative deamination of glutamate via GDH generates α-ketoglutarate, which enters into the Krebs cycle. The reaction is called as anaplerotic reaction, because it replenishes intermediates of the cycle that are used in biosynthetic processes. No extensive production of ammonia in peripheral tissue by GDH is observed. Thus, GDH contributes little to the blood level of ammonia, which is maintained at extremely low concentration (ammonia is higly toxic at concentration above 50 mole/L in blood). Therefore, ammonia is produced in the GDH reaction at sites where it is directly excreted as NH4+ (kidney), or incorporated in the non-toxic urea (liver). Reductive amination is carried out when α-ketoglutarate and ammonium ion are converted to glutamate by GDH with NAD(P)H/H+ consumption (Figure 1.). In mammals, the GDH equilibrium is shifted in favour of the production of ammonia and α-ketoglutarate (oxidative deamination) because of the high NAD/NADH ratio. The low rate of reductive amination probably relates also to the high Km of GDH for ammonia. Figure 1. Reaction catalyzed by GDH Isoenzymes Are Encoded by Different Genes Two isoforms of GDH have been identified in humans, GDH1 and GDH2, which are encoded by the genes GLUD1 and GLUD2, respectively. GLUD1 is expressed in many tissues with the highest expression in the liver, brain and pancreas cells, whereas GLUD2 is mainly expressed in retina, neural tissue, kidney (proximal tubules) and also in steroidogenic organs such as testes. The ratio of expression of the two genes is different in various tissues. For example, in liver only GDH1 is present, whereas in cortical neurons only GDH2 is expressed while; astrocytes contain comparable amounts of both isoenzymes. Both isoenzymes are present in the mitochondrial matrix of cells. GDH2 activity is low and therefore glutamate is mainly converted into α-ketoglutarate by GDH1. Enzyme kinetic properties of GDH1 In liver, GDH is present at much higher levels than in any other mammalian organs representing around 1% of all proteins expressed. The GDH is an ubiquitous enzyme showing the highest activity in the liver. The prevalent direction of the GDH reaction is determined by Km for substrates/coenzymes and also by the NAD/NADH or NADP+/NADPH ratios present in mitochondria. These different ratios are one of the key to keeping up the prevalent direction of the GDH reaction (forward reaction in Fig 1.). On the other hand, the Km values for glutamate (1.8 mM) and NH4+ (3.2 mM) are much higher than the Km values for α-ketoglutarate (0.7 mM). A very low affinity for ammonia (high Km of about 3.2 mM in liver and Km of about 18.3 mM in brain) and high mitochondrial NAD/NADH ratio determine the faster rate to reach equilibrium from the oxidative deamination side of the GDH reaction. GDH kinetics are different depending on the used coenzymes NAD+ or NADP+. While the Km values of GDH for glutamate are similar in reactions with NAD+ and NADP+ as coenzyme (Table 1.), the Vmax of GDH using NAD+ is 2.5-fold higher than that of with NADP+ in liver. GDH Kinetic parameters GDH + NAD+ GDH + NADP+ vmax (in mol/mL/minute) 1.26 0.51 Km (in mM for glutamate) 1.92 1.66 Table 1. Kinetic Parameters of GDH. Regulation of GDH 1. Post-Translational Modifications of GDH - Covalent modification In humans, the activity of GDH is controlled through mono-ADP-ribosylation, a covalent modification using NAD as substrate is carried out by the sirtuin 4 (SIRT4). By ADP- ribosylating GDH, SIRT4 inhibits its activity and blocks the conversion of glutamate (and glutamine, which is converted to glutamate in cells) to α-ketoglutarate, which can be used to provide energy by being used in the citric acid cycle to ultimately produce ATP. This regulation is relaxed in response to caloric restriction and low blood glucose. The control of GDH through mono-ADP-ribosylation is particularly important in insulin-producing β cells of pancrease. 2. Allosteric regulation GDH enzyme activity is allosterically inhibited by ATP and GTP and allosterically activated by ADP, GDP, and leucine. ENZYME KINETIC ASSAYS TO CHARACTERIZE THE L-GLUTAMATE DEHYDROGENASE ENZYME ACTIVITIES I.) NAD-SPECIFIC GDH (OXIDATIVE DEAMINATION) ACTIVITY Principle GDH catalyzes the reversible NAD-linked oxidative deamination of glutamate into α- ketoglutarate and ammonia. To explore GDH kinetics in vitro, in the first experiments we apply different concentrations of glutamate as a substrate in combination with the coenzyme NAD+ on the so called NAD-specific GDH activity. The time dependent appearance of NADH is measured at 340 nm by spectrophotometry, where the increase in the absorbance measured is proportional with the GDH activity. GDH L-Glutamate+NAD⁺+H₂O ► α-Ketoglutarate +NH₃+ NADH + H⁺ The appearance of NADH is measured at 340 nm by spectrophotometry. Reagents A. Buffer solution: 50 mM K-phosphate buffer, pH:7.4 containing 2,5 mM EGTA B. NAD⁺solution: 100 mM (dissolved in distilled water) C. L-Glutamate solution: 1 M and 0.05 M (dissolved in distilled water) D. GDH (GDH1, liver-type): (dissolved in 50 mM K-phosphate buffer, pH:8.3) Equipments and Tools • Recording spectrophotometer, able to measure at 340 nm, • semi-microcuvettes with a light path of 1 cm, • L-shaped stirring rod made of glass, or plastic (it should fit into the cuvettes, and be able to carry at least 30 µl of reagent solution), • Micropipettes with tips. Procedure 1. Prepare the following reaction mixtures in semi-microcuvettes (d=1.0cm): Reaction mixtures 1. 2. 3. 4. 5. Buffer solution (A) 945 l 935 l 915 l 945 l 935 l 100 mM NAD (B) 25 l 25 l 25 l 25 l 25 l 0.05 M L-Glutamate (C) 10 l 20 l 40 l 1 M L-Glutamate (C) 10 l 20 l Calculate the conc. of L- glutamate in the reaction mixtures The enzymatic reactions are initiated by adding the enzyme at last. Before that the absorbance of the cuvettes are set to 0.00 value and this way they serve as blanks. A reagent blank serves for the correction for the small amount of error in the assay results that would show up and derive from the reagents (NAD and L-glutamate) themselves displaying some absorbance at 340 nm. To initiate the reaction mix in 20 µl of the GDH enzyme solution (E) with a stirring rod and gently mix the samples to achieve homogeneity. Start measuring reaction time. 2. Record the increase in absorbance at 340 nm for 5 minutes in a spectrophotometer, and calculate the ΔA per minute from the initial linear portion of the curve (ΔA test). Time 1. 2. 3. 4. 5. (minute) 0 1 2 3 4 5 3. Calculations Reaction rate (v) can be calculated by using the following formula: ΔA/min ×Vt Reaction rate (v) (U/ml GDH) = 6.22×1.0×Vs Were: Vt: total volume (1ml) Vs: volume of the added enzyme also in ml (0.02ml) 6.22: millimolar extinction coefficient of NADH at 340nm (㎠/micromole) 1.0: light path length (cm) GLUT concentration A per 5 A per minute V (µmol/min/ml) (mM) minutes 1. 2. 3. 4. 5. 4. Graphical display of the results: Plot the values for substrate (L-glutamate) on the x- axis (abscissa) and the corresponding calculated values for V on the y-axis (ordinate). Estimate the values of apparent Km and Vmax. II.) NADP-SPECIFIC GDH (OXIDATIVE DEAMINATION) ACTIVITY Principle: In the second experimental set up we assay the NADP-specific GDH activity, where the enzymatic reaction is carried out applying glutamate as substrate in combination with NADP as coenzyme. GDH L-Glutamate+NADP⁺+H₂O ► α-Ketoglutarate +NH₃+ NADPH + H⁺ The formation of NADPH is measured at 340 nm by spectrophotometry. Reagents A. Buffer solution: 50 mM K-phosphate buffer, pH:7.4 containing 2,5 mM EGTA B. NADP⁺solution: 100 mM (dissolved in distilled water) C. L-Glutamate solution: 1 M (dissolved in distilled water) D. GDH (GDH1, liver-type) diluent: (dissolved in 50 mM K-phosphate buffer, pH:8.3) Procedure 1. Prepare the following reaction mixture in semi-microcuvette (d=1.0cm): Reaction mixtures 1. Buffer solution (A) 935 l 100 mM NADP+ (B) 25 l 1 M L-Glutamate (C) 20 l Calculate the conc. of L- glutamate in the reaction mixtures The above prepared reaction mixture serve as absorbance reference (reagent blank), therefore you must set the zero absorbance value of the photometer with these. The enzymatic reactions are initiated by adding the enzyme at last. To initiate the reaction mix in 20 µl of the GDH enzyme solution (E) with a stirring rod and gently mix the sample to achieve homogeneity. Start measuring reaction time. 2. Record the increase in absorbance at 340 nm for 5 minutes in a spectrophotometer, and calculate the ΔA per minute from the initial linear portion of the curve (ΔA test). Time 1. (minute) 0 1 2 3 4 5 3. Calculations Reaction rate (v) can be calculated by using the following formula: ΔA/min ×Vt Reaction rate (v) (U/ml GDH) = 6.22×1.0×Vs Were: Vt: total volume (1ml) Vs: volume of the added enzyme also in ml (0.02ml) 6.22: millimolar extinction coefficient of NADPH at 340nm (㎠/micromole) 1.0: light path length (cm) III.) NADH-SPECIFIC GDH (REDUCTIVE AMINATION) ACTIVITY Principle Finally, assaying NADH-specific GDH (reductive amination) activity is based on the fact that the disappearance of NADH, which is measured at 340 nm by spectrophotometry, when the enzyme assay is carried out using α-ketoglutarate, NH₃+, and NADH.