Clinical applications of plasmatic enzymes studies.



Measuring plasma enzymes activity is an important tool in diagnosis and monitoring of treatment.


Enzymes that have a physiological rol in blood, like coagulation enzymes, are present in plasma, but also can be found small quantities of enzymes that normally are present in tissues.


These quantities increase in some diseases of tissues and organs, since as a consequence of increased death cells or changes in cell membranes permeability, intracellular enzymes are released into plasma, giving clues about some organs diseases.


There is a strong association between the finding of an increase in plasma of particular enzymes, and the damage of organs that are rich in those enzymes.


A brief (and incomplete)  list of enzymes that have been useful as diagnostic tools, and its main diagnostic uses include:


Enzymes and Related Diseases


Acid Phosphatase .- Some Prostatic diseases

Alanine aminotransferase (ALT). -Liver, Heart diseases

Aldolase .- Some muscle diseases

Alkaline Phosphatase.- Liver and Bone diseases

Amylase.- Pancreatic diseases

Angiotensin-Converting Enzyme (ACE) .- Active Sarcoidosis

Aspartate aminotransferase (AST).- Heart, Liver diseases

Cholinestarase (pseudocholinestarase).- Acute organophosphorus poisoning

Creatin Kinase (CK or CPK) .- Heart, Muscle diseases

Gamma-Glutamyltransferase (GGT).- Liver disease, alcohol rehabilitation

Lactate Dehydrogenase (LDH).- Heart, Liver, Brain diseases

Lipase .-Pancreatitis

Lysozyme.- Some acute leukemias


More general information about the use of enzymes in diagnostic can be found here.


As discussed previously, some enzymes that catalyze the same reaction, have different structure. They are called Isoenzymes or Isozymes.  The diagnostic importance of  the determination of the different isoenzymes of an enzyme, has been discussed in a former post.


Some basic (biochemistry) comments about H1N1 virus.



This picture represents an Influenza virus.


Observe the capside and the core, containing nucleic acids associated to proteins.


The genome of the Influenza A virus is formed by 8 segments of negative sense RNA. Negative sense means that the molecule runs from 3’ to 5’, so it can not be translated to proteins. This type of virus needs a RNA polymerase for the transcription of the original viral RNA to RNA with positive sense (5’3’) that can function as mRNA.


Observe also in the viral surface two important glycoproteins: Haemagglutinin and Neuraminidase (Sialidase).


Hemagglutinin is a kind of lectin. Lectins are glycoproteins related to cellular recognition. Hemagglutinin in viral capside allows the virus to bind, in lungs and throat, with sialic acid residues (derived of neuraminic acid) that are part of the proteins in the epithelial cell surface. An endocytosis process allows the transport of the virus inside the cell and the eventual replication of the virus.


Once the viruses have been replicated inside the cell, they are excreted from the host cell inside a spherical phospholipid membrane that contains Hemagglutinin and Neuraminidase.  In the same say that happened at the beginning of the infection, the Hemagglutinin binds to sialic acid residues, but the Neuraminidase hydrolyses the glycoside linkage between Hemagglutinin and the Sialic Acid residues.


N-Acetyl-Neuraminic Acid (Sialic acid)

N-Acetyl-Neuraminic Acid (Sialic acid)


General Action of a Glycosidase (Neuraminidase is a Glycosidase)

General Action of a Glycosidase (Neuraminidase is a Glycosidase)


In the absence of the viral Neuraminidase, the new formed viruses would remain linked to the sialic acids residues in the cell surface or in the glycoproteins in the respiratory tract mucus, and they would get trapped and impeded of infecting other cells. 


Hemagglutinin (H) and Neuraminidase (N) also show antigenic properties and Influenza A viruses are classified in subtypes according to the kind of antibodies that these proteins raise. Sixteen subtypes of H (HA) and nine types of N (NA) are known. The Influenza virus H1N1 is then the Influenza A virus with Hemagglutinin subtype H1 and Neuraminidase subtype N1.


This picture, from the Influenza Laboratory of the CDC shows the Influenza A H1N1 virus.  (Compare the picture with the graphic shown at the beginning of this post).



The drugs Oseltamivir (Tamiflu®) and Zanamivir (Relenza ®) have been effective in the treatment of infections with H1N1 virus. These antiviral drugs are potent competitive inhibitors of Neuraminidase, particularly when they are used in the first 48 hours after illness onset, e.g., before the virus is able to release itself from the initial host cells and infect other cells.


Updated CDC recommendations about the use of these antiviral agents can be found in these links:


This graphic represent the structure of Oseltamivir



In fact, Oseltamivir is a prodrug (a precursor of the active drug), since the  carboxylic ester linkage is hydrolyzed in the liver, so Oseltamivir becomes  Oseltamivir carboxylate, the active form, with a conformation that looks like the natural substrate of the enzyme, the N-acetyl-neuraminic acid, a form of Sialic acid)



Updated information about Influenza A H1N1 virus, can be found at the CDC site


CDC: H1N1 Flu (Swine Flu)


This is the official site of the Mexican government with news and recommendations about the Influenza (in Spanish):

Induced fit model of Enzyme-Substrate interaction


Answer to Question E-05


b) Induced fit



The Induced fit model describes the formation of the E-S as a result of the interaction between the  substrate and a flexible active site.  The substrate produces changes in the conformation on the enzyme, aligning properly the groups in the enzyme. It allows  better binding and catalytic effects. 





This model opposes to the former Lock and Key model (c) , that explained the formation of the E-S complex as a result of the binding of complementary geometrical rigid structures, as a lock and a key.


Follow this link to find a very good animation that represents these two models:



The Concerted model (a) and the Sequential model (e) are models used to explain the allosteric changes of conformation of an enzyme from the T structure to the R structure and viceversa. In the concerted model all the subunits that form the allosteric protein change conformation at once, while in the sequential model the change in conformation of one subunit favors the change in conformation of the other subunits and so on.


The Michaellis Menten model (d) is related to the kinetics of enzyme catalyzed reactions, and describes the relationship between the concentration of substrate and enzyme velocity in a reaction where no allosteric effects exist.


More information about:


Michaelis Menten model:


Induced fit model:


Concerted Model and Sequential Model:





Isoenzymes or Isozymes


Answer to Enzyme Question E-04


Answer (b) isozymes


                                           Lactate Dehydrogenase LDH1  (MMMM)




                             Lactate Dehydrogenase (LDH M4)


Isozymes or Isoenzymes are proteins with different structure which catalyze the same reaction. Frequently they are oligomers made with different polypeptide chains, so they usually differ in regulatory mechanisms and in kinetic characteristics.


From the physiological point of view, isozymes allow the existence of similar enzymes with different characteristics, “customized” to specific tissue requirements or metabolic conditions.


One example of the advantages of having isoenzymes for adjusting the metabolism to different conditions and/ or in different organs is the following:


Glucokinase and Hexokinase are typical examples of isoenzymes. In fact, there are four Hexokinases: I, II, III and IV. Hexokinase I is present in all mammalian tissues, and Hexokinase IV, aka Glucokinase, is found mainly in liver, pancreas and brain.


Both enzymes catalyze the phosphorylation of Glucose:


Glucose + ATP —–à Glucose 6 (P) + ADP


Hexokinase I has a low Km and is inhibited by glucose 6 (P).  Glucokinase is not inhibited by Glucose 6 (P) and his Km is high. These two facts indicate that the activity of glucokinase depends on the availability of substrate and not on the demand of the product.


Since Glucokinase is not inhibited by glucose 6 phosphate, in conditions of high concentrations of glucose this enzyme continues phosphorylating glucose, which can be used for glycogen synthesis in liver. Additionally, since Glucokinase has a high Km, its activity does not compromise the supply of glucose to other organs; in other words, if Glucokinase had a low Km, and since it is not inhibited by its product, it would continue converting glucose to glucose 6 phosphate in the liver, making glucose unavailable for other organs (remember that after meals, glucose arrives first to the liver through the portal system).


Since isoenzymes have different tissue distributions, their study is an important tool in assessing the damage to specific organs.


Examples of the diagnostic use of isoenzymes are the study of Lactate Dehydrogenase and Creatine Kinase.



Lactate Dehydrogenase (LDH)


It is formed by the association of five peptide chains of two different kinds of monomers: M and H


The variants seen in humans are:


LDH1: M M M M (abundant in heart, brain erythrocytes; around 33% of serum LDH)


LDH2: M M M H (abundant in heart, brain erythrocytes; around 45% of serum LDH)


LDH3: M M H H (abundant in brain, kidneys, lung; around 18 % of serum LDH)


LDH4: M H H H ((abundant in liver, skeletal muscle, kidney; around 3% of serum LDH)


LDH5: H H H H ((abundant in liver, skeletal muscle, ileum; around 1 % of serum LDH)



In myocardial infarction, Total LDH increases, and since heart muscle contains more LDH1 than LDH2, LDH1 becomes greater than LDH2 between 12 and 24 hours, after the infarction, so the ratio LDH1/LDH2 becomes higher than 1 and will stay flipped for several days.


 An increase of LDH 5 in serum is seen in different hepatic pathologies: cirrhosis, hepatitis and others. An increase of LDH5 in heart diseases usually indicates secondary congestive liver involvement.


Creatine Kinase :



Creatine Kinase (CK) aka Creatine phosphokinase (CPK) is a similar example: three isoenzymes formed by combinations of different subunits:


CK1 (BB) is abundant in brain and smooth muscle (practically absent form serum)

CK2 (MB) is abundant in cardiac muscle, some in skeletal muscle (practically absent from serum)

CK3 (MM) is abundant in skeletal muscle and cardiac muscle (practically 100 % of serum CK)






                                                                       Creatine Kinase CK3



They can be differentiated based on their different electrophoretic mobility.


The primary clinical use of CK studies is the diagnosis of Myocardial Infarction,

(increased in the MB variant), but CK is also increased in different conditions

as muscular diseases and traumas (MM and MB) and brain trauma and brain surgery (BB). 


CK2 appears in serum within 6 hours after the myocardial infarction and is cleared after 24 to 48 hours. A persistence of CK2 in serum indicates extension of the infarction to other areas or another infarction.



For more information, please read:





The Medical Biochemistry page: Enzymes in the diagnosis of pathology.


Sacher, S. R and McPherson, R.A.:

Widmann’s Clinical Interpretation of laboratory Tests.

11th Edition. 2000 F.A Davis Company, Philadelphia, PA


Lactate Dehydrogenase


Creatine Kinase


Clinical Methods: The Clinical, Physical and Laboratory Investigations. Creatine Kinase



Zymogens or Proenzymes


Original Question


 Answer to E-02: (e) zymogens.






                                            Representation of a zymogen: Chymotrypsinogen


Zymogens or proenzymes are inactive precursors of enzymes. Observe that it is different than saying that they are “inactive enzymes”. An inactive enzyme is an enzyme that has lost its activity because of different factors, like physical factors, chemical factors or even metabolic factors. A zymogen is a molecule that needs to be activated in order to become an active enzyme, so it is more accurate to say that they are inactive precursors of enzymes, than to say that they are inactive enzymes.  Digestive enzymes, some coagulation factors and other proteins are synthesized as zymogens.


The synthesis of digestive enzymes in an inactivated form is a “safe” mechanism for the cells synthesizing some enzymes, since the proteolytic enzymes synthesized in this way are not activated until they abandon the cell.


Pepsin is synthesized as pepsinogen, trypsin is synthesized as trypsinogen, chymotripsin as chymotrypsinogen,  carboxypeptidase as procarboxypeptidase, and these zymogens are activated (usually when an external factor release an inhibitor peptide from their structure), only when they have been secreted in the gastrointestinal tract.


A good example of what occurs when some zymogens become active enzymes inside the cells is seen in acute pancreatitis , in which the premature activation of some of the pancreatic enzymes  like trypsine, phospholipase A2 and elastase, produce the autodigestion of pancreatic tissue.



Enzyme Specificity


Original Question (E-03)







Answer: ( c ) Relative specificity


One of the main characteristics of enzymes is their high specificity.


Enzymes are specific for:

a)     the substrate

b)     the reaction

 It means that they catalyze the transformation of  just one substrate or a family of substrates that are structurally related, catalyzing only one of the possible reactions of the substrate(s).


When the enzyme only can act on one substrate, it is said that the enzyme shows absolute specificity for the substrate. It is the case of succinate dehydrogenase, that is specific for succinate, or the L-glutamate dehydrogenase, specific for glutamate.


If the enzyme binds to some structurally related substrates, the enzyme shows relative specificity over substrate. L- amino acid Oxidase, for example can catalyze the oxidation of different amino acid of the L series, but not the oxidation of D- amino acids.


This characteristic of some enzymes is used with advantages in some clinical situations. For example, en patients intoxicated with methanol, ethanol is used in the treatment. The enzyme can bind to any of both alcohols (relative specificity), but has 10 to 20 more affinity for ethanol, so ethanol is metabolized instead of methanol.  Avoiding the oxidation of methanol for favoring its elimination without transformation, is very important, since the metabolic oxidation of methanol produces in the body the very dangerous formaldehide  and formic acid. (For an interesting review of Methanol intoxication, click here: Korabathina, K: Methanol)



The specificity of action is related to the fact that the enzyme only catalyzes one of the possible transformations of a substrate.


In the case of glutamate, for example,  it can experiment different transformations, but each of them requires a different enzyme, for example:


Glutamate to:


Glutamine (Fixation of ammonia) :      Glutamine synthase

GABA (Decarboxylation):                          Glutamate Decarboxyase     

Alfaketoglutarate:                                        Gutamate Dehydrogenase


The specificity of enzymes depends on the characteristics of the active site. It is the region of the enzyme where it binds to the substrate before the substrate transformations happens.


Interactive Concepts in Biochemistry: Enzyme Specificity




The binding of the enzyme to specific substrate(s) depends of different groups (usually lateral chains of amino acids) related to the active site:

a)     groups that form the backbone, that gives the appropriate conformation for the binding;

b)     the orientation groups, that “oblige” the substrate(s) to adopt the appropriate orientation for the reaction,

c)      the environmental groups, that give  the hydrophobic, polar or negative o positive environment necessary for warranting an appropriate affinity between the enzyme and the specific substrate, and

d)     the catalytic groups, responsible of creating the necessary “tensions” for breaking old linkages and forming new ones among the metabolites implied in the reaction: substrate(s), coenzymes or other prosthetic groups.   






A: About Enzymes (E-01)

Original question




Answer:  (b)


Kinases are a group of enzymes that catalyzes the interchange of phosphate groups between rich in energy phosphorylated compounds and diverse substrates.


These enzymes are very important:

a)     In increasing the energetic level of different compounds, converting them in metabolically active molecules.

b)     In the generation of ATP, GTP, in metabolic pathways.

c)      In the covalent modification of enzymes activity.


The typical kinase enzyme catalyzes the following reaction:


Substrate + ATP  ———-> Substrate-(P)  + ADP


Usually the common names of kinases are based on the substrate that is phosphorylated:


– Glucokinase: the substrate is glucose.


   Glucose + ATP ———–> Glucose 6 (P) + ADP


– Hexokinase: The substrate is a hexose (this enzyme is less specific than glucokinase)


   Hexose + ATP ————> Hexose (P) + ADP


– Phosphofructokinase (PFK): The substrate is fructose phosphate.


   Fructose 6 (P) + ATP —–> Fructose 1, 6 bis (P) + ADP            (PFK-1)

   Fructose 6 (P) + ATP——> Fructose 2, 6 bis (P)  + ADP            (PFK-2)


– Protein Kinase: The substrate is a Protein


  Protein + ATP ————-> Protein-(P) + ADP


– Tyrosine kinase:  The substrate is a residue of tyrosine in a protein (In a strict sense, tyrosine kinase is a kind of  Protein kinase).



In some cases, ATP or GTP are formed by the transfer to ADP or GDP of a phosphate of the substrate, which is linked through a rich in energy bond.



The reaction of pyruvate kinase:

Phosphoenol pyruvate +ADP——-> Pyruvate + ATP


Reaction of phosphoglycerokinase (This is an exception among kinases, since this reaction is reversible)

1,3 bisphosphoglycerate + ADP< ——–> 3 Phosphoglycerate + ATP



Besides the importance of kinases in activating metabolites and generating ATP, the phosphorylation of enzymes and other proteins through the action of kinases is a general mechanism of metabolic control at a molecular level.


The activity of several enzymes is controlled by covalent modification based on the addition or remotion of Phosphate groups. Some enzymes increase their activity when phosphorylated, while in other enzymes the activity is decreased. The opposite is valid also for the dephosphorylation of enzymes: while some of them increase the activity when dephosphorylated, others decrease the activity.


Protein Kinases (enzymes that phosphorylate proteins) and Protein Phosphatases (enzymes that release phosphates from proteins) are responsible of maintaining the appropriate equilibrium in several metabolic processes.


A very clear example of this process is seen in the regulation of glycogen metabolism:


Protein kinases (Phosphorylase kinase and Protein Kinase A) provoke phosphorylation of Glycogen phosphorylase and Glycogen Synthase respectively. As a consequence, the activity of Glycogen Phosphorylase increases and the activity of Glycogen Synthase decreases. Global Result: Increase in the conversion of glycogen to glucose.


Phosphatases provoke the release of the phosphates from Glycogen Phosphorylase and from Glycogen Synthase. As a consequence, the activity of Glycogen Phosphorylase decreases and the activity of Glycogen Synthase increases. Global result: An increase in the incorporation of glucose to glycogen.


Particular interest shows the activity of Tyrosine Kinase.


This enzyme is part of a receptor-enzyme molecule, which is activated by the interaction of the part of the molecule that acts as receptor, with some hormones, like insulin, and growth factors. The enzyme then autophosphorylates the tyrosine residues in the enzymatic part of the molecule, gaining in activity and phosphorylating other proteins, like IRSs.


In summary, Kinases, through the catalysis of the interchange of phosphate groups between rich in energy phosphorylated compounds and diverse substrates facilitate very different metabolic process of great relevance for the organism.