Glycerol Enters Glycolysis at the Level of What Intermediate

Glycerol Kinase

The presence of glycerol kinase allows glycerol released via the hydrolysis of stored TAG to be converted to glycerol phosphate which, in turn, allows reincorporation of released FA, or of FA derived from exogenous lipoproteins, into storage TAG, albeit at an energetic cost to the adipocyte.

From: Encyclopedia of Biological Chemistry (Second Edition) , 2013

Gluconeogenesis

Larry R. Engelking , in Textbook of Veterinary Physiological Chemistry (Third Edition), 2015

Glycerol

Since glycerol kinase activity is absent in white adipose tissue, glycerol becomes a waste product of lipolysis, and is converted to glucose in the liver, and to a lesser extent in the kidney. This substrate becomes a significant source of glucose in hibernating animals (e.g., the black bear), where lipolysis becomes necessary for survival (see Chapter 76). It should be noted that the synthesis of glucose from glycerol requires fewer steps (and therefore less energy), than synthesis from other precursors. Glycerol utilization in gluconeogenesis also bypasses the dicarboxylic acid (DCA) shuttle (see pyruvate carboxylase below), thereby allowing oxaloacetate (OAA) to be reutilized in the TCA cycle.

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Role of Mitochondria in Adipose Tissues Metabolism and Plasticity

Audrey Carrière , Louis Casteilla , in Mitochondria in Obesity and Type 2 Diabetes, 2019

3.2 Glyceroneogenesis

Because of low glycerol kinase activity—even if its expression is induced by rosiglitazone in murine and human white adipocytes ^{82, 83} —glyceroneogenesis is the main source of glycerol in white adipocytes. ^{79, 84} In the fed state, glycerol 3-phosphate is produced from the reduction of dihydroxyacetone phosphate (from glycolysis or the pentose phosphate pathway) by the glycerol-3-phosphate dehydrogenase activity. During fasting, precursors other than glucose can be metabolized to form glycerol-3-phosphate such as pyruvate, lactate, and some amino acids. One of the rate-limiting steps in glyceroneogenesis is the synthesis of phosphoenolpyruvate from oxaloacetate, which is catalyzed by the phosphoenolpyruvate carboxykinase (PEPCK), present both in cytosol and mitochondria. PEPCK protein content is much greater in BAT than in WAT, maybe because the primary function of BAT is thermogenesis, which is fueled by beta-oxidation of fatty acids supplied by the triacylglycerol/fatty acid futile cycle (an ATP-consuming process involved in thermogenesis). In addition, BAT exhibits important glycerol kinase activity that enables direct recycling of glycerol produced by hydrolysis of stored triacylglycerol into glycerol-3-phosphate. ⁸⁵ In addition to providing ATP, mitochondria support glyceroneogenesis by providing tricarboxylic acid cycle intermediates such as pyruvate and malate that feed cytosolic oxaloacetate by malate transport into cytosol. ⁷⁹

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Enzyme Nanoarchitectures: Enzymes Armored with Graphene

Neelam Yadav , ... Chandra S. Pundir , in Methods in Enzymology, 2018

5.2 Biosensor Based on Immobilization of Nanoparticles of Lipase, Glycerol Kinase, and Glycerol-3-Phosphate Oxidase

The porcine pancreas lipase, glycerol kinase (GK) from Cellulomonas sp., and glycerol-3-phosphate oxidase (GPO) from Aerococcus viridans were prepared by the above desolvation method (Pundir & Aggarwal, 2017) (Fig. 11). These NPs were characterized by TEM and FTIR and immobilized onto Au electrode. The performance of the biosensor was maximum at pH 6.5 at 35°C. The working potential of biosensor was 1.2   V and response time 5   s. Biosensor was evaluated in terms of linear concentration range of 1.0–100 and 100–500   mg   dL^{−   1}, respectively. The detection limit was 1.0   μg   mL^{−   1}, and the analytical recovery of added concentration was 95% and 96% at 50 and 100   mg   dL^{−   1}, respectively. The coefficients of variation were 2% and 2%, respectively. The storage stability of working electrode was 90 days.

Fig. 11. Schematic representation of attachment of functionalized lipase, glycerol kinase (GK), and glycerol-3-phosphate oxidase (GPO) NPs and their electrochemical reaction onto Au electrode (Pundir & Aggarwal, 2017).

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Conceptual Background and Bioenergetic/Mitochondrial Aspects of Oncometabolism

Simone Patergnani , ... Paolo Pinton , in Methods in Enzymology, 2014

3 Monitoring Intracellular ATP Using Alternative Enzymatic Methods

The main advantage of the luciferin–luciferase assay is the very rapid detection of ATP levels, but this technique requires a luminometer. Should a luminometer not be available, it is possible to use alternative procedures that take advantage of standard enzymatic methods. Two alternative enzymatic methods for ATP measurement are proposed below.

The first method is based on the glycerokinase/glycerolphosphate oxidase/horseradish peroxidase-coupled assay. Briefly, the assay is composed of a three-step enzymatic reaction:

1.

ATP (from a measured sample)   +   glycerol   +   glycerokinase   →   glycerol-3-phosphate   +   ADP

2.

Glycerol-3-phosphate   +   O₂  +   glycerolphosphate oxidase   →   phosphodioxyacetone   +   H₂O₂

3.

H₂O₂  +   TMPD (TetraMethylPhenyleneDiamine)   +   horseradish peroxidase   →   H₂O   +   TMPD*

*The absorbance of oxidized TMPD is measured spectrophotometrically at 610   nm.

This method was originally proposed for the detection of triglycerides rather than ATP.

The second method is based on the hexokinase/glucose-6-phosphate dehydrogenase-coupled assay. This method is the most common and was originally used for glucose detection. After some modification, it was also used for hexokinase detection in subfractionated mitochondrial protein complexes, as described by Beutner, Ruck, Riede, Welte, and Brdiczka (1996) or by Wieckowski, Brdiczka, and Wojtczak (2000) and Wieckowski et al. (2001). Briefly, the assay is composed of a two-step enzymatic reaction:

1.

ATP   +   glucose   +   hexokinase   →   glucose-6-phosphate   +   ADP

2.

Glucose-6-phosphate   +   NADP   +   glucose-6-phosphate dehydrogenase   →   6-phosphogluconate   +   NADPH

The absorbance of reduced NADPH can be measured spectrophotometrically at 340   nm or fluorimetrically at an excitation/emission of 340/450   nm, respectively.

Below, we include an accurate description of this tool and provide a precise standardization of the method for quantifying the amount of ATP.

3.1 Reagent setup

1.

Reaction buffer: 100   mM TEA (triethanolamine), 16   mM MgSO₄ (magnesium sulfate) and 10   mM EDTA (ethylenediaminetetraacetic acid disodium salt dehydrate); pH 7.6

2.

Stock solutions: glucose (1   M stock), NAD (1   M stock), ATP (0.75   M stock), hexokinase (0.5   U/ml stock), and glucose-6-phosphate dehydrogenase (200   U/ml stock)

3.2 Sample preparation

1.

Resuspend the cells in a small amount of PBS (depending on the pellet size).

2.

Add TCA (trichloroacetic acid) to a final concentration of 2%. The TCA should contain 0.001% of xylenol blue dye as a pH indicator. A red color indicates an acidic pH.

3.

After the TCA extraction, the sample should be diluted to 0.1% (concentration of TCA).

4.

Neutralize the sample using the Tris–acetate buffer, pH 7.7. The color of the neutralized sample should change from red to yellow. If necessary, add more of the Tris–acetate buffer, pH 7.7.

5.

Keep the sample on ice and use it for ATP measurements or store it at −   80  ^°C.

An alternative method has been described by Manfredi, Yang, Gajewski, and Mattiazzi (2002).

1.

Resuspend the cells in a small amount of PBS (depending on the pellet size).

2.

Add 10   μl of ice-cold 0.4 M perchloric acid per milligram of protein.

3.

Keep the suspension on ice for 30   min, then centrifuge it at 14,000   rpm for 10   min at 4   °C.

4.

Collect the supernatant and neutralize it with 10   μl of 4 M K₂CO₃ per 100   μl of supernatant.

5.

Keep the suspension on ice for 10   min and then at −   80   °C for 1–2   h to precipitate the perchlorate.

6.

Centrifuge the sample again at 14,000   rpm for 10   min at 4   °C.

7.

Keep the collected supernatant on ice and use it for ATP measurements. Alternatively, store it at −   80   °C.

3.3 Measurements

1.

Dispense 2.5   ml of the reaction buffer into the cuvette and add the following:

–

10   μl of 1 M glucose

–

10   μl of 1 M NAD⁺

–

20   μl of glucose-6-phosphate dehydrogenase (200   U/ml stock)

–

25–50   μl of the sample

2.

Record the fluorescence trace. Begin the reaction with the addition of 20   μl of hexokinase (0.5   U/ml stock) and wait for the plateau. If the change in fluorescence is small, add more sample. To ensure that the amount of hexokinase in the reaction was not limiting, it is possible to add an additional hexokinase to the cuvette. No significant change in the fluorescence should be observed (see Fig. 16.3).

Figure 16.3. Representative trace of an ATP measurement in Zajdela tumor cell lysates. The data were obtained from 50   μl of Zajdela tumor cell lysates. HK, hexokinase; ml, milliliter; AU, arbitrary unit.

3.4 Standardization of the method

After reaching the plateau, add small volumes of the ATP stock solution (diluted to the proper concentration, if necessary). Measure the increase in fluorescence (see Fig. 16.3). It is important to perform this standardization on the same day of the experiment (e.g., for each ATP stock and sample buffer).

3.5 Notes

The protocol presented above (in Section 3) is useful for measuring the total cellular ATP level. It is very difficult to distinguish between the cytosolic and mitochondrial ATP levels using this method. These difficulties are based on the practical impossibility of separating the cytosolic fraction without damaging the mitochondria (during mild homogenization or plasma membrane permeabilization using digitonin). Mitochondrial damage will cause the release of ATP from broken mitochondria and contaminate the cytosolic ATP pool. Recently, Soccio et al. described a fast method for isolating cytosol-enriched fractions in which mitochondrial integrity is preserved by using an isotonic grinding medium (Soccio, Laus, Trono, & Pastore, 2013). This fluorimetric method to assay ATP content is practically identical to the one presented in the section above (Section 3).

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Rational Design of Enzyme-Nanomaterials

V. Aggarwal , C.S. Pundir , in Methods in Enzymology, 2016

4.1 Combined Assay of Mixture of Free/Native Lipase, GK and GPO

4.1.1 Preparation of Mixture of Enzymes

Materials : Lipase (from porcine pancreas), glycerol kinase (GK, from Cellulomonas), glycerol-3-phosphate oxidase (GPO, from Aerococcus viridans), MgCl₂, ATP, potassium ferrocyanide, 4-aminophenazone, 3,5 dichloro-2-hydroxybenzene sulfonic acid (DHBS), triolein solution, and Triton X-100.

Equipment: UV–vis spectrophotometer.

Dissolve 2.0   mg of lipase (from porcine pancreas; 40–70   U/mg)   +   1.0   mg GK (from Cellulomonas sp; 25–75   U/mg)   +   0.1   mg GPO (from Aerococcus viridans; 113   U/mg) into 1.0   ml of 0.02 M sodium phosphate buffer (PB), pH 7.0, of 50–100   units of each of the three enzymes in the mixture and store at 4   °C until use.

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Dry Chemistry

In Laboratory Techniques in Biochemistry and Molecular Biology, 1993

Creatine kinase (CK)

Principle: Creatine kinase is activated by N-acetylcysteine within 80 s.

$\begin{array}{c}\text{Creatine phosphate}+\text{ADP}\stackrel{\text{CK}}{\rightleftharpoons }\text{creatine}+\text{ATP}\\ \text{Glycerol}+\text{ATP}\stackrel{\text{glycerol}\text{kinase}}{⟶}\text{glycerol}-3-\text{phosphate}+\text{ADP}\\ {\text{Glycerol}-3-\text{phosphate}+\text{O}}_{\text{2}}\stackrel{\text{glycerophosphate}\text{oxidase}}{⟶}\text{dihydroxyacetone}{\text{phosphate}+\text{H}}_{\text{2}}{\text{O}}_{\text{2}}\\ {\text{H}}_{\text{2}}{\text{O}}_{\text{2}}+{\text{indicator}}_{(\text{reduced})}\stackrel{\text{peroxidase}}{⟶}{\text{indicator}}_{(\text{oxidized})}{+\text{H}}_{\text{2}}\text{O}\end{array}$

Structure of the reagent carrier:

The test area contains per cm²:

Buffer	not declared
Creatine phosphate	116	μg
Glycerol kinase	≥ 1.0	U
Glycerol	4.4	μg
Glycerophosphate oxidase	≥ 0.09	U
Peroxidase	≥ 1.7	U
Ascorbate oxidase	≥ 0.41	U
ADP	4	μg
Diadenine pentaphosphate	1	μg
Diarylimidazole indicator	9.4	μg
N-acetylcysteine	11.3	μg
EGTA	16	μg

Measurement wavelength: 642 nm.

Duration of measurement: Approx. 3 min.

Sample material: Blood, heparin blood, serum or heparin plasma.

Range of measurement:	24 – approx. 2 400 U/l (37°C)
	15.1– approx. 1 500 U/l (30°C)
	9.8– approx. 1 000 U/l (25°C)

Reference interval: Data applicable for the sample material blood, serum and plasma

	37°C	30°C	25°C
Females	24–170 U/l	15–110 U/l	0–70 U/l
Males	24–195 U/l	15–130 U/l	0–80 U/l

Conversion factors:	U/l (25°C) = 0.41 · U/l (37°C)
	U/l (30°C) = 0.63 · U/l (37°C)

Storage life of the reagent carrier: At temperatures between +2°C and +30°C stable up to the imprinted date of expiry if stored in closed containers.

Interferences:

Drug or metabolite in sample	Concentration up to which no interference occurred [mg/l]	Concentration usually appearing in serum [mg/l]	Interference, direction [mg/l]	Clinically relevant
Acetylsalicylic acid	1 000	20–100, occ. 300	−	−
Ampicillin	1 000	5, occ. 320	−	−
Ascorbic acid	20	6.5–17.5	> 20↓	no
Bezafibrate	100	4–13	−	−
Bilirubin	60 mg/dl	0.2–1.0 mg/dl	−
Caffeine	200	2–10, occ. up to 60	−	−
Calcium dobesilate	20	6–68, occ. 70	> 20↓	yes
Carbocromen	30	0.8–2.4	−	−
Chloramphenicol	200	up to 22	−	−
Dipyridamole	100	up to 0.6	−	−
Furosemide	1 750	12, occ. 50	−	−
Glibenclamide	1	0.1–0.2	−	−
Haemoglobin	10 g/1	< 0.025 g/1	−
Indomethacin	100	0.3–6.0	−	−
Intralipid	10 000	?	−	?
Methyldopa	100	2, occ. 7	−	−
Nicotinic acid	400	4–10	−	−
Nitrofurantoin	16	1.8–5.5	−	−
Noramidopyrine	200	not detectable	−	−
Oxytetracycline	160	1.5–2.4, occ. 21	−	−
Paracetamol	200	5–20	−	−
Phenprocoumon	20	0.2–3.6	−	−
Phenazopyridine	25	?	−	?
Phenytoin	200	5–20	−	−
Probenecid	1 000	100–200	−	−
Procaine	2	up to 2.7	−	−
Pyridamol	100	?	−	?
Pyritinol	20	?	−	?
Quinidine	60	2–5	−
Sulfa-methoxazole	80	2.5–60, occ. up to 125	> 80↓	yes
Theophylline	200	10–20	−	−
Triglycerides	1 400 mg/di	< 200 mg/dl	−
Trimethoprim	18	1–3, occ. 5	−	−

Calcium ions and myokinase are not considered to be disturbing substances.

References: R171, RN16, manufacturers' method description [September 1989], evaluation report.

Influence of the volume of the sample: No data have been published.

Influence of a change in starting time: No data have been published.

Influence of the haematocrit value (R171): Haematocrit values between 25 and 50% do not influence the measurement.

Statistical data from evaluations:

–

Intra-assay imprecision

–

Inter-assay imprecision

Correlation data to comparative methods:

Straight line equation	Correlation coefficient	Number of samples compared	Comparative method	References
y = 1.00x+ 0.38	−	−	CK-NAC (37°C)	R49
y = 1.00x – 7.89	0.997	59	CK-NAC (37°C)	R171
y = 0.98 x − 2.6	0.996	93	CK-NAC (37°C)	a
y = 1.08 x − 10.7	0.988	125	CK-NAC (37°C)	a
y = 0.91 x − 0.91	0.996	100	CK-NAC (37°C)	a
y = 1.06 x − 9.9	0.983	−	CK-NAC (37°C)	RN9
y = 1.03 x − 10.2	0.995	−	CK-NAC (37°C)	RN10
y = 0.89 x + 3.7	0.962	166	Ektachem	RN16
Reflotron vs Reflotron whole blood (y) vs serum (x)
y = 1.07 x − 11.2	0.988	47		RN9
plasma (y) vs serum (x)
y = 1.01x – 2.4	0.994	47		RN9

a

Evaluation report Boehringer Mannheim.

Further reference: RN13.

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Dry Chemistry

In Laboratory Techniques in Biochemistry and Molecular Biology, 1993

Triglycerides

Principle:

$\begin{array}{l}\text{Triglycerides}\stackrel{\text{lipase},\text{pH}8}{⟶}\text{glycerol}+\text{free}\text{fatty}\text{acids}\\ \text{Glycerol}+\text{ATP}\stackrel{\text{glycerol}\text{kinase},{\text{Mg}}^{2+}}{⟶}\text{glycerol}-3-\text{phosphate}+\text{ADP}\\ \text{Glycerol}-3-\text{phosphate}+\text{NAD}\stackrel{\begin{array}{l}\text{glycerol}-3-\text{phosphate}-\\ \text{dehydrogenase}\end{array}}{⟶}\text{dehydroxyacetone}-3-\\ \text{phosphate}+\text{NADH}\\ \text{NADH}+\text{INT}\stackrel{\text{diaphorase}}{⟶}{\text{NAD}}^{+}+\text{INTH}\end{array}$

INT = 2-(p- indophenyl)- 3-(p-nitrophenyl)-5- phenyltetrazolium chloride, oxidized formazan dye

INTH = reduced formazan dye

During an incubation and reaction phase amounting to a total of 240 seconds the change of reflectance between the 60th and the 240th second at a wave-length of 580 nm is recorded. The result can be calculated by means of the calibration data that have been set up beforehand.

Composition of the reagent carrier (proportionate mass percentage ω):

1.2%

Lipase (microbial, 3 600 U/mg)

0.5%

Glycerol-kinase (microbial, 60 U/mg)

1.1%

Glycerol-3-phosphate-dehydrogenase (rabbit, 110 U/mg)

1.4%

Diaphorase (microbial, 35 U/mg)

7.3%

NAD

3.0%

ATP, disodium salt

1.6%

Magnesium sulfate heptahydrate

5.0%

2-(p-indophenyl)-3(p-nitrophenyl)-5-phenyltetrazolium chloride

78.9%

Non-reactive components

Storage and storage life of the reagent carrier: Temperatures below 30°C are recommended for storage. Do not store in refrigerator or deep-freezer. Since the reagent carriers are sensitive to light and moisture, they must be protected from direct sunlight and humidity. After the first opening of the container the reagent carriers must be used within 120 days.

Stability of calibration: 7 days.

Sample material: Serum, EDTA plasma or heparin plasma.

Dilution of samples: 1 part sample + 8 parts de-ionised water.

Range of measurement: 40–500 mg/dl or 0.45–5.65 mmol/l.

Reference interval: 20–180 mg/dl or 0.23–2.05 mmol/l.

Interferences:

Drug or metabolite in sample	Concentration up to which no interference occurred [mg/l]	Concentration usually appearing in serum [mg/l]	Interference, direction [mg/l]	Clinically relevant
Ascorbic acid	200	up to 18	−	−
Bilirubin	10 mg/dl	up to 1.2 mg/dl	−
Glucose	1 000 mg/dl	70–110 mg/dl	−
Haemoglobin	0.3 mg/dl	<0.025 g/1	> 0.3 g/l↑
Lactate dehydrogenase	750 U/l (37oC)	100–225 U/1 (37°C)	−
Urea-N	60 mg/dl	8–23 mg/d1	−

Statistical data from evaluations:

–

Intra-assay imprecision (S54, S125)

Mean value [mg/dl]	Coefficient of variation [%]
66	5.9
130	3.6
200	3.9
253	3.3
400	4.3

–

Inter-assay imprecision (S54, S125)

Mean value [mg/dl]	Coefficient of variation [%]
66	6.8
130	2.8
200	3.6
253	2.3
400	5.3

Correlation data to comparative methods:

Straight line equation	Correlation coefficient	Number of samples compared	Comparative method	References
y = 0.98 x + 2.8	0.99	78	UV method	S125
y = 0.96 x ± 0	0.98	266	−	S197
y = 0.95 x + 4.6	0.988	77	GPO-PAP	S181

Further references: S196, S243.

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Hypoglycemia

Dorit Koren , Andrew Palladino , in Genetic Diagnosis of Endocrine Disorders (Second Edition), 2016

Glycerol Metabolism Disorder

As discussed previously, lipolysis of triacylglycerols yields free fatty acids and glycerol. Glycerol enters the bloodstream and subsequently is taken up by either liver or kidneys. In these organs (primarily in the liver), glycerol is converted into glycerol-3-phosphate in a reaction catalyzed by glycerol kinase. Hepatic glycerol-3-phosphate is then oxidized into dihydroxyacetone phosphate (DHAP) in a reversible redox reaction catalyzed by glycerol-3-phosphate dehydrogenase; DHAP can then enter either the glycolytic or gluconeogenic pathways.

The main clinical disorder of glycerol metabolism that has been described is glycerol kinase deficiency. Glycerol kinase is encoded by the GK gene on chromosome Xp21.2; thus, in distinction to all of the fatty acid oxidation defects, it is an X-linked rather than autosomal disorder. Symptoms are due to the inability for glycerol to enter the gluconeogenic pathway; GK deficiency is a very rare disorder and has the following phenotypic presentations: ^177,184

1.

Complex GK deficiency: This infantile-onset form involves deletion not only of the GK gene, but also of the gene causing Duchenne muscular dystrophy (DMD) and/or the gene causing hypoplasia adrenal congenita (AHC). Thus, it has a very severe presentation, either involving progressive muscular dystrophy or glucocorticoid and mineralocorticoid insufficiency. This form is characterized by severe developmental delay.

2.

Isolated GK deficiency: This form involves isolated GK deficiency, without concomitant DMD or AHC. It is further subdivided into two phenotypes:

a.

Juvenile-onset GK deficiency: Affected children may present with severe hypoglycemic episodes accompanied by profound metabolic acidosis, vomiting, lethargy, and hypotonia, episodes triggered by the same catabolic stressors as described earlier – fasting, illness, or prolonged exercise. As fasting tolerance increases with age due to increased hepatic gluconeogenic capacity from nonglycerol precursors, symptoms often disappear.

b.

Adult-onset GK deficiency: Individuals with this form of GK deficiency are asymptomatic; this condition is often detected incidentally.

All forms of GK deficiency are characterized by pseudohypertriglyceridemia, since the elevated glycerol is detected by assays as triglycerides. Levels may be 40 times above the normal range.

Once the pseudohypertriglyceridemia is detected, definitive diagnosis is based upon detection of the GK mutation by sequence analysis. In individuals with the infantile and juvenile-onset forms, treatment is based upon avoidance of fasting combined with a low-fat, high-carbohydrate diet, and treatment of any metabolic exacerbations as discussed earlier with fatty acid oxidation disorders. Those with DMD or AHC are treated as per standard for those disorders. Individuals with adult-onset GK deficiency do not require treatment.

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Lipid Metabolism

Antonio Blanco , Gustavo Blanco , in Medical Biochemistry, 2017

Fat metabolism

TAGs must be totally hydrolyzed before they can be used by tissues. Much of this hydrolysis affects the fat stored in adipose tissue. Thus, there is a permanent degradation, or lipolysis, of the stored TAGs that is catalyzed by intracellular lipases, whose activity is regulated to suit the body needs. The products formed (FAs and glycerol) are released into the plasma, where FAs bind to albumin.

Exogenous TAGs transported by chylomicrons and the endogenous ones transported by VLDLs are hydrolyzed by LpL in capillaries and the released FAs enter the cells where they are used. Hydrolyzed fats, either from stored TAG or transported by lipoproteins, also release free glycerol, which is taken up by cells that can metabolize it.

Glycerol Metabolism

Before it can be used by cells, glycerol requires its previous activation by phosphorylation, which is why only tissues possessing glycerokinase are able to metabolize free glycerol. Glycerokinase is found in liver, kidney, intestine, and lactating mammary gland tissues. It catalyzes the conversion of glycerol into l-glycerol-3-phosphate, by transferring a phosphate group from ATP. The reaction is practically irreversible.

Glycerol-3-phosphate is converted into dihydroxyacetone, a triose phosphate of the glycolysis pathway, by the action of the NAD-linked glycerophosphate dehydrogenase. As noted when considering the stages of glycolysis, dihydroxyacetone is subsequently converted into glyceraldehyde-3-phosphate in the reaction catalyzed by phosphotriose isomerase.

Both reactions are reversible and through the same steps, glycerol-3-phosphate can be obtained from triose phosphate. The formation of triose phosphate provides a pathway that leads to the complete degradation of glycerol in the glycolysis and the citric acid cycles. Moreover, it can also follow the gluconeogenic pathway to form glucose or glycogen. Glycerol-3-phosphate is an important metabolite in TAGs and glycerophospholipids synthesis.

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Regulation of Carbon Assimilation in Bacteria

J. Plumbridge , in Encyclopedia of Microbiology (Third Edition), 2009

PTS Regulation of Glycerol Kinase

The use of glycerol is also regulated by PTS-mediated phosphorylation in B. subtilis . The phosphorylated target is not a transcription factor but an enzyme, the glycerol kinase, encoded by glpK. PEP-dependent phosphorylation of GlpK by HPr on a histidine residue increases the kinase activity about tenfold, thus enhancing use of glycerol and formation of glycerol-3P. The presence of glucose promotes the dephosphorylation of GlpK (via a reversal of the reaction with HPr) and exerts a form of catabolite repression by reducing the activity of GlpK and hence the concentration of Gly3P. Gly3P is the inducing signal for the GlpP antiterminator protein that controls the glpFK operon, encoding the glycerol facilitator transport protein and the kinase, so that the end result is that in the presence of glucose the glpFK operon is not induced. However, in the absence of glucose or another preferred PTS carbon source and the presence of glycerol, the activity of GlpK is high, Gly3P concentrations are high, and the glpFK operon is derepressed.

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Glycerol Enters Glycolysis at the Level of What Intermediate

Source: https://www.sciencedirect.com/topics/medicine-and-dentistry/glycerol-kinase

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