Re:transamination의 효소 aspartate transaminase(AST), alanine aminotransferase(ALT)의 이해
작성자문형철작성시간20.04.13조회수786 목록 댓글 0beyond reason
드디어 GOT(AST)에 대한 이해에 도달함
간, 심장, 근육, 신장, 뇌, 적혈구에 존재하는 transamination 효소 aspartate aminotransferase(AST)
즉 간에 존재하는 단백질(아미노산) degradation과정에 중요한 효소.
ALT는 alanine aminotransferase임.
AST is similar to alanine transaminase (ALT) in that both enzymes are associated with liver parenchymal cells. The difference is that ALT is found predominantly in the liver, with clinically negligible quantities found in the kidneys, heart, and skeletal muscle, while AST is found in the liver, heart (cardiac muscle), skeletal muscle, kidneys, brain, and red blood cells.
Aspartate transaminase
Aspartate transaminase (AST) or aspartate aminotransferase, also known as AspAT/ASAT/AAT or (serum) glutamic oxaloacetic transaminase (GOT, SGOT), is a pyridoxal phosphate (PLP)-dependent transaminase enzyme (EC 2.6.1.1) that was first described by Arthur Karmen and colleagues in 1954.[2][3][4] AST catalyzes the reversible transfer of an α-amino group between aspartate and glutamate and, as such, is an important enzyme in amino acid metabolism. AST is found in the liver, heart, skeletal muscle, kidneys, brain, and red blood cells. Serum AST level, serum ALT (alanine transaminase) level, and their ratio (AST/ALT ratio) are commonly measured clinically as biomarkers for liver health. The tests are part of blood panels.
Function[edit]
Aspartate transaminase catalyzes the interconversion of aspartate and α-ketoglutarate to oxaloacetate and glutamate.
L-Aspartate (Asp) + α-ketoglutarate ↔ oxaloacetate + L-glutamate (Glu)
As a prototypical transaminase, AST relies on PLP (Vitamin B6) as a cofactor to transfer the amino group from aspartate or glutamate to the corresponding ketoacid. In the process, the cofactor shuttles between PLP and the pyridoxamine phosphate (PMP) form.[5] The amino group transfer catalyzed by this enzyme is crucial in both amino acid degradation and biosynthesis. In amino acid degradation, following the conversion of α-ketoglutarate to glutamate, glutamate subsequently undergoes oxidative deamination to form ammonium ions, which are excreted as urea. In the reverse reaction, aspartate may be synthesized from oxaloacetate, which is a key intermediate in the citric acid cycle.[6]
Isoenzymes[edit]
Two isoenzymes are present in a wide variety of eukaryotes. In humans:
- GOT1/cAST, the cytosolic isoenzyme derives mainly from red blood cells and heart.
- GOT2/mAST, the mitochondrial isoenzyme is present predominantly in liver.
These isoenzymes are thought to have evolved from a common ancestral AST via gene duplication, and they share a sequence homology of approximately 45%.[7]
AST has also been found in a number of microorganisms, including E. coli, H. mediterranei,[8] and T. thermophilus.[9] In E. coli, the enzyme is encoded by the aspCgene and has also been shown to exhibit the activity of an aromatic-amino-acid transaminase (EC 2.6.1.57).[10]
Structure[edit]
X-ray crystallography studies have been performed to determine the structure of aspartate transaminase from various sources, including chicken mitochondria,[11] pig heart cytosol,[12] and E. coli.[13][14] Overall, the three-dimensional polypeptide structure for all species is quite similar. AST is dimeric, consisting of two identical subunits, each with approximately 400 amino acid residues and a molecular weight of approximately 45 kD.[7] Each subunit is composed of a large and a small domain, as well as a third domain consisting of the N-terminal residues 3-14; these few residues form a strand, which links and stabilizes the two subunits of the dimer. The large domain, which includes residues 48-325, binds the PLP cofactor via an aldimine linkage to the ε-amino group of Lys258. Other residues in this domain – Asp 222 and Tyr 225 – also interact with PLP via hydrogen bonding. The small domain consists of residues 15-47 and 326-410 and represents a flexible region that shifts the enzyme from an "open" to a "closed" conformation upon substrate binding.[11][14][15]
The two independent active sites are positioned near the interface between the two domains. Within each active site, a couple arginine residues are responsible for the enzyme’s specificity for dicarboxylic acid substrates: Arg386 interacts with the substrate’s proximal (α-)carboxylate group, while Arg292 complexes with the distal (side-chain) carboxylate.[11][14]
In terms of secondary structure, AST contains both α and β elements. Each domain has a central sheet of β-strands with α-helices packed on either side.
Mechanism[edit]
Aspartate transaminase, as with all transaminases, operates via dual substrate recognition; that is, it is able to recognize and selectively bind two amino acids (Asp and Glu) with different side-chains.[16] In either case, the transaminase reaction consists of two similar half-reactions that constitute what is referred to as a ping-pong mechanism. In the first half-reaction, amino acid 1 (e.g., L-Asp) reacts with the enzyme-PLP complex to generate ketoacid 1 (oxaloacetate) and the modified enzyme-PMP. In the second half-reaction, ketoacid 2 (α-ketoglutarate) reacts with enzyme-PMP to produce amino acid 2 (L-Glu), regenerating the original enzyme-PLP in the process. Formation of a racemic product (D-Glu) is very rare.[17]
The specific steps for the half-reaction of Enzyme-PLP + aspartate ⇌ Enzyme-PMP + oxaloacetate are as follows (see figure); the other half-reaction (not shown) proceeds in the reverse manner, with α-ketoglutarate as the substrate.[5][6]
- Internal aldimine formation: First, the ε-amino group of Lys258 forms a Schiff base linkage with the aldehyde carbon to generate an internal aldimine.
- Transaldimination: The internal aldimine then becomes an external aldimine when the ε-amino group of Lys258 is displaced by the amino group of aspartate. This transaldimination reaction occurs via a nucleophilic attack by the deprotonated amino group of Asp and proceeds through a tetrahedral intermediate. As this point, the carboxylate groups of Asp are stabilized by the guanidinium groups of the enzyme’s Arg386 and Arg 292 residues.
- Quinonoid formation: The hydrogen attached to the a-carbon of Asp is then abstracted (Lys258 is thought to be the proton acceptor) to form a quinonoid intermediate.
- Ketimine formation: The quinonoid is reprotonated, but now at the aldehyde carbon, to form the ketimine intermediate.
- Ketimine hydrolysis: Finally, the ketimine is hydrolyzed to form PMP and oxaloacetate.
This mechanism is thought to have multiple partially rate-determining steps.[18] However, it has been shown that the substrate binding step (transaldimination) drives the catalytic reaction forward.[19]
Clinical significance[edit]
AST is similar to alanine transaminase (ALT) in that both enzymes are associated with liver parenchymal cells. The difference is that ALT is found predominantly in the liver, with clinically negligible quantities found in the kidneys, heart, and skeletal muscle, while AST is found in the liver, heart (cardiac muscle), skeletal muscle, kidneys, brain, and red blood cells.[20] As a result, ALT is a more specific indicator of liver inflammation than AST, as AST may be elevated also in diseases affecting other organs, such as myocardial infarction, acute pancreatitis, acute hemolytic anemia, severe burns, acute renal disease, musculoskeletal diseases, and trauma.[21]
AST was defined as a biochemical marker for the diagnosis of acute myocardial infarction in 1954. However, the use of AST for such a diagnosis is now redundant and has been superseded by the cardiac troponins.[22]
AST is commonly measured clinically as a part of diagnostic liver function tests, to determine liver health. However, it is important to keep in mind that the source of AST (and, to a lesser extent, ALT) in blood tests may reflect pathology in organs other than the liver. In fact, when the AST is higher than ALT, a muscle source of these enzymes should be considered. For example, muscle inflammation due to dermatomyositis may cause AST>ALT. This is a good reminder that AST and ALT are not good measures of liver function because they do not reliably reflect the synthetic ability of the liver and they may come from tissues other than liver (such as muscle).
Laboratory tests should always be interpreted using the reference range from the laboratory that performed the test. Example reference ranges are shown below:
| Patient type | Reference ranges[23] |
| Male | 8–40 IU/L |
| Female | 6–34 IU/L |
Alanine transaminase
Alanine transaminase (ALT) is a transaminase enzyme (EC 2.6.1.2). It is also called alanine aminotransferase (ALAT) and was formerly called serum glutamate-pyruvate transaminase (SGPT) or serum glutamic-pyruvic transaminase (SGPT) and was first characterized in the mid-1950s by Arthur Karmen and colleagues.[1] ALT is found in plasma and in various body tissues but is most common in the liver. It catalyzes the two parts of the alanine cycle. Serum ALT level, serum AST (aspartate transaminase) level, and their ratio (AST/ALT ratio) are commonly measured clinically as biomarkers for liver health. The tests are part of blood panels.
Function[edit]
ALT catalyzes the transfer of an amino group from L-alanine to α-ketoglutarate, the products of this reversible transamination reaction being pyruvate and L-glutamate.
ALT (and all aminotransferases) require the coenzyme pyridoxal phosphate, which is converted into pyridoxamine in the first phase of the reaction, when an amino acid is converted into a keto acid.
Clinical significance[edit]
ALT is commonly measured clinically as part of liver function tests and is a component of the AST/ALT ratio.[2] When used in diagnostics, it is almost always measured in international units/liter (IU/L)[3] or µkat. While sources vary on specific reference range values for patients, 0-40 IU/L is the standard reference range for experimental studies.[2]
Elevated levels[edit]
Test results should always be interpreted using the reference range from the laboratory that produced the result. However typical reference intervals for ALT are:
| Patient type | Reference ranges[4] |
| Female | ≤ 34 IU/L |
| Male | ≤ 45 IU/L |
Significantly elevated levels of ALT (SGPT) often suggest the existence of other medical problems such as viral hepatitis, diabetes, congestive heart failure, liver damage, bile duct problems, infectious mononucleosis, or myopathy, so ALT is commonly used as a way of screening for liver problems. Elevated ALT may also be caused by dietary choline deficiency. However, elevated levels of ALT do not automatically mean that medical problems exist. Fluctuation of ALT levels is normal over the course of the day, and they can also increase in response to strenuous physical exercise.[5]
When elevated ALT levels are found in the blood, the possible underlying causes can be further narrowed down by measuring other enzymes. For example, elevated ALT levels due to hepatocyte damage can be distinguished from bile duct problems by measuring alkaline phosphatase. Also, myopathy-related elevations in ALT should be suspected when the aspartate transaminase (AST) is greater than ALT; the possibility of muscle disease causing elevations in liver tests can be further explored by measuring muscle enzymes, including creatine kinase. Many drugs may elevate ALT levels, including zileuton, omega-3 acid ethyl esters (Lovaza),[6] anti-inflammatory drugs, antibiotics, cholesterol medications, some antipsychotics such as risperidone, and anticonvulsants.[citation needed] Paracetamol (acetaminophen) may also elevate ALT levels.[7]
For years, the American Red Cross used ALT testing as part of the battery of tests to ensure the safety of its blood supply by deferring donors with elevated ALT levels. The intent was to identify donors potentially infected with hepatitis C because no specific test for that disease was available at the time. Prior to July 1992, widespread blood donation testing in the USA for hepatitis C was not carried out by major blood banks. With the introduction of second-generation ELISA antibody tests for hepatitis C, the Red Cross changed the ALT policy. As of July 2003, donors previously disqualified for elevated ALT levels and no other reason may be reinstated as donors when they contact the donor-counseling department of their regional Red Cross organization.[8]
In 2000, the American Association for Clinical Chemistry determined that the appropriate terminology for AST and ALT are aspartate aminotransferase and alanine aminotransferase. The term transaminase is outdated and no longer used in liver disease.[9]