Serine dehydratase

Serine dehydratase or L-serine ammonia lyase (SDH) is in the β-family of pyridoxal phosphate-dependent (PLP) enzymes. SDH is found widely in nature, but its structure and properties vary among species. SDH is found in yeast, bacteria, and the cytoplasm of mammalian hepatocytes. SDH catalyzes the deamination of L-serine to yield pyruvate, with the release of ammonia.^[1]

This enzyme has one substrate, L-serine, and two products, pyruvate and NH₃, and uses one cofactor, pyridoxal phosphate (PLP). The enzyme's main role is in gluconeogenesis in the liver's cytoplasm.^{[citation needed]}

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In this webcast, we're gonna take a look at one step of the shikimate pathway. We'll focus in on this elimination reaction catalyzed by dehydroquinate dehydratase. You're not really gonna learn anything new in this webcast, but what this allows us to do is to apply some of the principles that you've been hearing about in the previous webcasts to one particular step of this reaction mechanism. So you know that when we talked about elimination reactions, we were interested in knowing what the stereochemistry of that elimination was. Does it proceed by syn or anti and that allows us to potentially rule out some mechanisms by which this reaction may, ah, may go, and so that's exactly what we're gonna do. We're gonna look first at the stereochemistry and then we'll take a look at the mechanism. So of the two hydrogens that could possibly be lost, the pro-S or the pro-R hydrogen across this carbon-carbon single bond to make this carbon-carbon double bond, deuterium studies show experimentally that it's the pro-R hydrogen that’s removed, the hydrogen_S remains behind. That tells us right away that what we're dealing with is a syn elimination. This is pretty easy to pick up in these ring compounds. These ring compounds we see the transformation of that, ah, ring system which divides basically the plane of what will become the carbon-carbon double bond, and so we can see that H_R, the proton that is removed, and water both have stereochemistry that is down below that plane and so we can imagine that the elimination is on the same side, the groups that leave are the same side of the plane of what becomes the double bond, that would be the syn elimination. But let me take you through the process in full detail. Any time we're dealing with a ring compound, um, you ought to be able to transform it from these planar structures into the two chair structures. Remember that anytime we draw, um, a six-membered ring we have to think about both conformations that are available. And what we're gonna learn in this, um, example when we work through both ring flipped forms, that both of them show it's a syn elimination process. So in the one-ring flip form, the hydroxyl group would be drawn in the, ah, axial conformation, and then here it is in the equatorial conformation. The pro-R hydrogen, the one that's removed, here's it's in the equatorial and in the ring flip form, it's in the axial. In both cases when we look down the carbon-carbon bond, which will become the carbon-carbon double bond, ah, and draw that Newman projection so the plane of what will become the double bond is drawn there, and you can see that H_R and OH are on the same side, that's the syn elimination pathway. Similarly, in this other ring flip form we get the exact same answer as we must, but we need to go through that process and convince our self of that. Looking down the carbon-carbon bond that becomes the carbon-carbon double bond, H_R and OH are on the same side of that, what will become the plane of that double bond. Again, we conclude it's a syn elimination. Now, with ring compounds, because there isn't this conformational flexibility, it's really quite easy to pick this up and it's a little bit more challenging in these open chain conformations where you have to find the right conformation that proceeds by the elimination. So since it's a syn elimination, we can conclude that the mechanism is not E2 and we can guess that the mechanism might be an E1cb mechanism. And that's basically what goes on, but in dehydroquinate dehydratase, there is a lysine present in the active site, and we can imagine that the combination, now, of a carbonyl on the lysine, um, is what we expect to produce the, by covalent catalysis or nucleophilic catalysis, um, the iminium ion intermediate. And indeed, that's how this reaction proceeds. Ah, it's an E1cb that involves the iminium ion formation. So the pattern that you should get used to when you see in the active site a lysine with a carbonyl substrate is to think about the iminium ion formation. Remember that that's going to enhance the acidity of any carbon, ah, that is α to the, um, carbon-nitrogen double bond, so that α position to the iminium ion has enhanced acidity, H_R then can be removed by some side chain, which at this point it's not disclosed what it is, but there's a basic side chain that then becomes protonated in the enzyme active site and we have the enamine structure drawn here. So, the carbon-nitrogen single bond connected to a carbon-carbon double bond and the, what was the base, has now been protonated. That's useful because the bond that is going to be broken, we're going to lose the elements of water and we can do that with general acid catalyzed, ah, process using the hydrogen, using the proton from the conjugate acid of the base that we used in the step preceding, so that's how the electron flow looks for that loss of water, um. And overall, this two-step process of deprotonation followed by β elimination, this second step is really best labeled as a β elimination, so E1cb is the two-step process, the second step of these being the β elimination which goes on here, to cause the loss of water, and uh, the iminium ion will be regenerated. Hydrolysis of that iminium ion, ah, that is we're going to just add water into that and it follows similar to what we saw with the aldolase, ah, enzyme produces the final product, which is the, um, dehydroshikimate proc, pro, product that's shown here. Let's take a look at one more thing. This, if you notice, I've drawn, um, this structure in kind of a funny way. Ah, here is the normal chair form, but I've drawn this in a twist-boat form, and it turns out there's a reason that people think that the enzyme forces the substrate to occupy this twist-boat conformation because it, as you'll see on the next slide, changes the orientation of this hydrogen bound to carbon in the α position to be almost perpendicular to the plane of that double bond. By inducing that conformation in the substrate, the relationship between the carbon-hydrogen bond and the π system is to place, um, those groups such that there can be overlap, orbital overlap between the CH σ bond and the π* system. So here's π* of the iminium group and with that conformation, there's really, ah, excellent overlap between the σ orbital and the π* orbital, which also enhances the acidity. So, somehow, and we saw this with strain in a, when we talked about one of the, ah, the thermolysin, ah, peptidase, ah, enzyme, ah, basically strain here is used again, in this case to enhance orbital overlap, σ to π* orbital overlap, making that α hydrogen even more acidic.

Nomenclature

Serine Dehydratase is also known as:^[2]

L-serine ammonia-lyase
Serine deaminase
L-hydroxyaminoacid dehydratase
L-serine deaminase
L-serine dehydratase
L-serine hydro-lyase

Structure

The holoenzyme SDH contains 319 residues, one PLP cofactor molecule.^[1] The overall fold of the monomer is very similar to that of other PLP-dependent enzymes of the Beta-family. The enzyme contains a large catalytic domain that binds PLP and a small domain. The domains are linked by two residues 32-35 and 138-146, with the internal gap created being the space for the active site^[1]

Cofactor Binding

The PLP cofactor is positioned in between the Beta-strands 7 and 10 of the large domain and lies on the large internal gap made between small and large domain. The cofactor is covalently bonded through a Schiff base linkage to Lys41. The cofactor is sandwiched between the side chain of Phe40 and the main chain of Ala222. Each of the polar substituents of PLP is coordinated by functional groups: the pyridinium nitrogen of PLP is hydrogen-bonded to the side chain of Cys303, the C3-hydroxyl group of PLP is hydrogen-bonded to the side chain of Asn67, and the phosphate group of PLP is coordinated by main chain amides from the tetraglycine loop.^[1]^[3] (Figure 3 and Figure 4).

Mechanism

The reaction catalyzed by serine dehydratase follows the pattern seen by other PLP-dependent reactions. A Schiff base linkage is made and the aminoacrylate group is released which undergoes non-enzymatic hydrolytic deamination to pyruvate.^[4]

Inhibitors

According to the series of assays performed by Cleland (1967), the linear rate of pyruvate formation at various concentrations of inhibitors demonstrated that L-cysteine and D-serine competitively inhibit the enzyme SDH.^[5] The reason that SDH activity is inhibited by L-cysteine is because an inorganic sulfur is created from L-Cysteine via Cystine Desulfrase and sulfur-containing groups are known to promote inhibition.^[6] L-threonine competitively inhibits Serine Dehydratase as well.

Moreover, insulin is known to accelerate glycolysis and repress induction of liver serine dehydratase in adult diabetic rats.^[7] Studies have been conducted to show insulin causes a 40-50% inhibition of the induction serine dehydratase by glucagon in hepatocytes of rats.^[8] Studies have also shown that insulin and epinephrine inhibit Serine Dehydratase activity by inhibiting transcription of the SDH gene in the hepatocytes.^[9] Similarly, increasing levels of glucagon, increase the activity of SDH because this hormone up-regulates the SDH enzyme. This makes sense in the context of gluconeogenesis. The main role of SDH is to create pyruvate that can be converted into free glucose. And glucagon gives the signal to repress gluconeogenesis and increase the amount of free glucose in the blood by releasing glycogen stores from the liver.

Homocysteine, a compound that SDH combines with Serine to create cystathionine, also noncompetitively inhibits the action of SDH. Studies have shown that homocysteine reacts with SDH's PLP coenzyme to create a complex. This complex is devoid of coenzyme activity and SDH is not able to function (See Enzyme Mechanism Section).^[10] In general, homocysteine is an amino acid and metabolite of methionine; increased levels of homocysteine can lead to homocystinuria(see section Disease Relevance).^[11]

Biological function

In general, SDH levels decrease with increasing mammalian size.^[12]

SDH enzyme plays an important role in gluconeogenesis. Activity is augmented by high-protein diets and starvation. During periods of low carbohydrates, serine is converted into pyruvate via SDH. This pyruvate enters the mitochondria where it can be converted into oxaloacetate, and, thus, glucose.^[13]

Little is known about the properties and the function of human SDH because human liver has low SDH activity. In a study done by Yoshida and Kikuchi, routes of glycine breakdown were measured. Glycine can be converted into serine and either become pyruvate via serine dehydratase or undergo oxidative cleavage into methylene-THF, ammonia, and carbon dioxide. Results showed the secondary importance of the SDH pathway.^[13]^[14]

Disease relevance

SDH may be significant in the development of hyperglycemia and tumors.

Nonketotic hyperglycemia is due to the deficiency of threonine dehydratase, a close relative of serine dehydratase. Serine dehydratase has also been found to be absent in human colon carcinoma and rat sarcoma. The observed enzyme imbalance in these tumors shows that an increased capacity for the synthesis of serine is coupled to its utilization for nucleotide biosynthesis as a part of the commitment to cellular replication in cancer cells. This pattern is found in sarcomas and carcinomas, and in tumors of human and rodent origin.^[15]

Evolution

Human and rat serine dehydratase cDNA are identical except for a 36 amino acid residue stretch. Similarities have also been shown between yeast and E. coli threonine dehydratase and human serine dehydratase. Human SDH shows sequence homology of 27% with the yeast enzyme and 27% with the E. coli enzyme.^[16] Overall PLP enzymes exhibit high conservation of the active site residues.^[16]

External links

Serine+dehydratase at the U.S. National Library of Medicine Medical Subject Headings (MeSH)

v t e Carbon–nitrogen lyases (EC 4.3)
4.3.1: ammonia-lyases	Histidine ammonia-lyase Formiminotransferase cyclodeaminase Serine dehydratase Phenylalanine ammonia-lyase
4.3.2: amidine-lyases	Argininosuccinate lyase Adenylosuccinate lyase

Metabolism: Protein metabolism, synthesis and catabolism enzymes

Essential amino acids are in Capitals

K→acetyl-CoA

LYSINE→	Saccharopine dehydrogenase Glutaryl-CoA dehydrogenase
LEUCINE→	3-Hydroxybutyryl-CoA dehydrogenase Branched-chain amino acid aminotransferase Branched-chain alpha-keto acid dehydrogenase complex Enoyl-CoA hydratase HMG-CoA lyase HMG-CoA reductase Isovaleryl coenzyme A dehydrogenase α-Ketoisocaproate dioxygenase Leucine 2,3-aminomutase Methylcrotonyl-CoA carboxylase Methylglutaconyl-CoA hydratase (See Template:Leucine metabolism in humans – this diagram does not include the pathway for β-leucine synthesis via leucine 2,3-aminomutase)
TRYPTOPHAN→	Indoleamine 2,3-dioxygenase/Tryptophan 2,3-dioxygenase Arylformamidase Kynureninase 3-hydroxyanthranilate oxidase Aminocarboxymuconate-semialdehyde decarboxylase Aminomuconate-semialdehyde dehydrogenase
PHENYLALANINE→tyrosine→	(see below)

G→pyruvate
→citrate

glycine→serine→	Serine hydroxymethyltransferase Serine dehydratase glycine→creatine: Guanidinoacetate N-methyltransferase Creatine kinase
alanine→	Alanine transaminase
cysteine→	D-cysteine desulfhydrase
threonine→	L-threonine dehydrogenase

G→glutamate→
α-ketoglutarate

HISTIDINE→	Histidine ammonia-lyase Urocanate hydratase Formiminotransferase cyclodeaminase
proline→	Proline oxidase Pyrroline-5-carboxylate reductase 1-Pyrroline-5-carboxylate dehydrogenase/ALDH4A1 PYCR1
arginine→	Ornithine aminotransferase Ornithine decarboxylase Agmatinase
→alpha-ketoglutarate→TCA	Glutamate dehydrogenase
Other	cysteine+glutamate→glutathione: Gamma-glutamylcysteine synthetase Glutathione synthetase Gamma-glutamyl transpeptidase glutamate→glutamine: Glutamine synthetase Glutaminase

G→propionyl-CoA→
succinyl-CoA

VALINE→	Branched-chain amino acid aminotransferase Branched-chain alpha-keto acid dehydrogenase complex Enoyl-CoA hydratase 3-hydroxyisobutyryl-CoA hydrolase 3-hydroxyisobutyrate dehydrogenase Methylmalonate semialdehyde dehydrogenase
ISOLEUCINE→	Branched-chain amino acid aminotransferase Branched-chain alpha-keto acid dehydrogenase complex 3-hydroxy-2-methylbutyryl-CoA dehydrogenase
METHIONINE→	generation of homocysteine: Methionine adenosyltransferase Adenosylhomocysteinase regeneration of methionine: Methionine synthase/Homocysteine methyltransferase Betaine-homocysteine methyltransferase conversion to cysteine: Cystathionine beta synthase Cystathionine gamma-lyase
THREONINE→	Threonine aldolase
→succinyl-CoA→TCA	Propionyl-CoA carboxylase Methylmalonyl CoA epimerase Methylmalonyl-CoA mutase

G→fumarate

PHENYLALANINE→tyrosine→	Phenylalanine hydroxylase Tyrosine aminotransferase 4-Hydroxyphenylpyruvate dioxygenase Homogentisate 1,2-dioxygenase Fumarylacetoacetate hydrolase tyrosine→melanin: Tyrosinase

G→oxaloacetate

asparagine→aspartate→	Asparaginase/Asparagine synthetase Aspartate transaminase

References

^ ^a ^b ^c ^d Sun L, Bartlam M, Liu Y, Pang H, Rao Z (March 2005). "Crystal structure of the pyridoxal-5'-phosphate-dependent serine dehydratase from human liver". Protein Science. 14 (3): 791–8. doi:10.1110/ps.041179105. PMC 2279282. PMID 15689518.
^ "KEGG ENZYME Database Entry". Kyoto Encyclopedia of Genes and Genomes. Kanehisa Laboratories. Retrieved 17 May 2011.
^ Toyota CG, Berthold CL, Gruez A, Jónsson S, Lindqvist Y, Cambillau C, Richards NG (April 2008). "Differential substrate specificity and kinetic behavior of Escherichia coli YfdW and Oxalobacter formigenes formyl coenzyme A transferase". Journal of Bacteriology. 190 (7): 2556–64. doi:10.1128/JB.01823-07. PMC 2293189. PMID 18245280.
^ Yamada T, Komoto J, Takata Y, Ogawa H, Pitot HC, Takusagawa F (November 2003). "Crystal structure of serine dehydratase from rat liver". Biochemistry. 42 (44): 12854–65. doi:10.1021/bi035324p. PMID 14596599.
^ Gannon F, Bridgeland ES, Jones KM (February 1977). "L-serine dehydratase from Arthrobacter globiformis". The Biochemical Journal. 161 (2): 345–55. doi:10.1042/bj1610345. PMC 1164512. PMID 322657.
^ Nakagawa H, Kimura H (November 1969). "The properties of crystalline serine dehydratase of rat liver". Journal of Biochemistry. 66 (5): 669–83. doi:10.1093/oxfordjournals.jbchem.a129180. PMID 5358627.
^ Freedland RA, Taylor AR (December 1964). "Studies on Glucose-6-Phosphatase and Glutaminase in Rat Liver and Kidney". Biochimica et Biophysica Acta (BBA) - Specialized Section on Enzymological Subjects. 92 (3): 567–71. doi:10.1016/0926-6569(64)90016-1. PMID 14264889.
^ Miura S, Nakagawa H (October 1970). "Studies on the molecular basis of development of serine dehydratase in rat liver". Journal of Biochemistry. 68 (4): 543–8. doi:10.1093/oxfordjournals.jbchem.a129384. PMID 5488777.
^ Kanamoto R, Su Y, Pitot HC (August 1991). "Effects of glucose, insulin, and cAMP on transcription of the serine dehydratase gene in rat liver". Archives of Biochemistry and Biophysics. 288 (2): 562–6. doi:10.1016/0003-9861(91)90236-C. PMID 1654838.
^ Pestaña A, Sandoval IV, Sols A (October 1971). "Inhibition by homocysteine of serine dehydratase and other pyridoxal 5'-phosphate enzymes of the rat through cofactor blockage". Archives of Biochemistry and Biophysics. 146 (2): 373–9. doi:10.1016/0003-9861(71)90139-1. PMID 4398884.
^ Hurd RW, Hammond EJ, Wilder BJ (March 1981). "Homocysteine induced convulsions: enhancement by vitamin B6 and inhibition by hydrazine". Brain Research. 209 (1): 250–4. doi:10.1016/0006-8993(81)91190-2. PMID 6260308. S2CID 29790535.
^ Rowsell EV, Carnie JA, Wahbi SD, Al-Tai AH, Rowsell KV (1979). "L-serine dehydratase and L-serine-pyruvate aminotransferase activities in different animal species". Comparative Biochemistry and Physiology. B, Comparative Biochemistry. 63 (4): 543–55. doi:10.1016/0305-0491(79)90061-0. PMID 318433.
^ ^a ^b Snell K (1984). "Enzymes of serine metabolism in normal, developing and neoplastic rat tissues". Advances in Enzyme Regulation. 22: 325–400. doi:10.1016/0065-2571(84)90021-9. PMID 6089514.
^ Koyata H, Hiraga K (February 1991). "The glycine cleavage system: structure of a cDNA encoding human H-protein, and partial characterization of its gene in patients with hyperglycinemias". American Journal of Human Genetics. 48 (2): 351–61. PMC 1683031. PMID 1671321.
^ Snell K, Natsumeda Y, Eble JN, Glover JL, Weber G (January 1988). "Enzymic imbalance in serine metabolism in human colon carcinoma and rat sarcoma". British Journal of Cancer. 57 (1): 87–90. doi:10.1038/bjc.1988.15. PMC 2246686. PMID 3126791.
^ ^a ^b Ogawa H, Gomi T, Konishi K, Date T, Nakashima H, Nose K, Matsuda Y, Peraino C, Pitot HC, Fujioka M (September 1989). "Human liver serine dehydratase. cDNA cloning and sequence homology with hydroxyamino acid dehydratases from other sources". The Journal of Biological Chemistry. 264 (27): 15818–23. doi:10.1016/S0021-9258(18)71550-0. PMID 2674117.

This page was last edited on 6 October 2023, at 05:28

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