Examples of enzymes working in reverse?

Examples of enzymes working in reverse?

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I have always been taught that enzymes can catalyze both the forward and reverse reaction, and will increase the reaction rate in both directions. I understand that the thermodynamics of the reaction are not altered by the enzyme, but I have yet to find a good answer / example for an enzyme that actually does this. (E.g. can a nuclease join DNA together if the products are in excess? Can a protease form a peptide bond?) I am having difficulty finding a resource online that actually justifies this claim without just saying "Yes enzymes work both ways."

Enzymes alter the rate of a reaction by lowering activation energy; they have no effect on the reaction equilibrium ($ce{K_{eq}}$). Since $ce{K_{eq}=frac{k_f}{k_r}}$ and $ce{K_{eq}}$ is constant, an increase in forward rate ($ce{k_f}$) requires a corresponding increase in the reverse rate ($ce{k_r}$). Intuitively it may help to think that the same effect an enzyme has on lowering the energy of the transition state in the forward direction should also be present in the reverse direction. Whether or not the reaction actually proceeds in reverse depends on the free energy difference between the reactants and products and their concentrations (among other things). See this answer for a great explanation.

Because their reactions are in equilibrium ($Delta Gapprox0,ce{K_{eq}approx1}$), many enzymes in the glycolytic pathway also catalyze the reverse reaction in gluconeogenesis.

[image source]

What about the ATP synthase? it uses proton flow to generate ATP but it can also burn ATP to generate proton flow.

Like other enzymes, the activity of F1FO ATP synthase is reversible. Large-enough quantities of ATP cause it to create a transmembrane proton gradient, this is used by fermenting bacteria that do not have an electron transport chain, but rather hydrolyze ATP to make a proton gradient, which they use to drive flagella and the transport of nutrients into the cell.

In respiring bacteria under physiological conditions, ATP synthase, in general, runs in the opposite direction, creating ATP while using the proton motive force created by the electron transport chain as a source of energy. The overall process of creating energy in this fashion is termed oxidative phosphorylation. The same process takes place in the mitochondria, where ATP synthase is located in the inner mitochondrial membrane and the F1-part projects into mitochondrial matrix. The consumption of ATP by ATP-synthase pumps proton cations into the matrix.

Examples of enzymes working in reverse?

Except three enzymes of Glycolysis (Hexokinase, PFK-I and Pyruvate kinase) all catalyse reversible reactions.

As these enzymes catalyse the backward reactions too they are part of Gluconeogenesis pathway.

(A comparison between the two pathways)

You mention nucleases and proteases, but if you turn these processes around and think about the actual nucleic acid or protein synthesis reactions an interesting point emerges:

These synthetic processes involve the production of pyrophosphate - not orthophosphate - from ATP (etc.).

(In the case of nucleic acid synthesis this should be obvious; in the case of protein synthesis I am refering to the aminoacyl-tRNA synthetase 'amino acid activation' reaction which drives peptide bond formation.)

Why pyrophosphate? The answer commonly given is that this is to prevent the reversal of the reaction. The generation of pyrophosphate achieves this because it is rapidly degraded to orthophosophate by pyrophosphatases in the cell. (See discussion of DNA polymerization in Berg et al.)

If this argument is accepted, it paradoxically illustrates the potential reversibility of the synthetic reactions.

Viral Tools for In Vitro Manipulations of Nucleic Acids

Boriana Marintcheva , in Harnessing the Power of Viruses , 2018 Reverse Transcriptases

Reverse transcriptases (RTs) are RNA dependent DNA pols initially isolated from retroviruses. In addition, RTs are coded by dsRNA viruses that utilize reverse transcription such as hepatitis B virus (replication of hepatitis is discussed in Chapter 1 ) and various retroelements in eukaryotes and prokaryotes. The enzyme telomerase maintaining the ends of the eukaryotic chromosomes is technically also a reverse transcriptase, although its mechanism is very distinct from conventional RTs. Historically, the discovery of RT revolutionized molecular biology leading to the revision of the central dogma and enabling scientists to develop new research tools that heavily influenced cloning, analysis of gene expression and RNA biology. HIV RT is one of the most extensively studied polymerases in the context of understanding the biology of this devastating virus and designing RT inhibitors as drugs to manage HIV infections. RTs exhibit three key enzyme activities ( Fig. 2.13 ): (1) RNA-dependent DNA pol that uses ssRNA template and a primer (tRNA Lys for HIV RT) to synthesize ssDNA/cDNA, which remains hybridized to its RNA template (2) RNAse H endonuclease, which selectively degrades the RNA strand of DNA/RNA hybrids and (3) DNA dependent DNA polymerase activity, converting the single-stranded cDNA into dsDNA. Conventional RT enzymes have two active sites, one executing the polymerase activities and another executing the endonuclease activity. RT are monomeric or dimeric proteins and some lack intrinsic RNAse H activity. The RTs of Moloney murine leukemia virus (M-MLV) and Avian myeloblastosis virus (AMV) are most frequently used as molecular tools in RT-PCR, RT-qPCR, cDNA cloning, RNA sequencing and any other experimental technique/approach that requires conversion of RNA to DNA. Site-directed mutagenesis and protein evolution have been utilized to optimize those enzymes improving thermostability and modulating RNAseH activity. Using thermostable version of RT is beneficial for lowering the nonspecific nucleic acid amplification and minimizing impact of complex secondary structures. Robust RNAseH activity is an advantage in RT-PCR, whereas lower RNAseH activity is beneficial in cDNA cloning protocols, especially when very long mRNA transcripts are reverse transcribed. In some cases RT is used only to produce the RNA/DNA hybrid and a conventional DNA pol carries out the cDNA to dsDNA polymerization step.

Figure 2.13 . Reverse transcriptase activities and mechanism of action.

Amino Acids and Polarity

What makes an amino acid polar or nonpolar? What level of polarity affects an amino acid's hydrophobicity or hydrophilic characteristics? This is the basic structure of an amino acid:

Notice that the basic structure always carries an amine group (NH2), and a carboxyl functional group (CO2H). The general formula for an amino acid is H2NCHRCOOH, which denotes the order in which the hydrogen and carbon atoms are bonded. The 20 amino acids have this same general structure. What makes them different is the R-linked side chain. Images of the 20 different amino acids.

Amino acids differ in their electronegativity in the R groups, causing differences in their hydrophobicity. The side chains denote whether an amino acid is:

Recall that the more electronegative an R side chain is compared with its amine and carboxyl, the more polar the amino acid. In general, side chains with hydrocarbon alkyl groups (CnHn), or benzene rings are non-polar. Examples: phenylalinine, Leucine, Isoleucine The number of alkyl groups affects the polarity. The more CnHn groups, the more nonpolar.

What makes an amino acid more polar?

  • Acids, amides, amines, and alcohols make an amino acid more polar.
  • Acids (the Rs can be Hydrogens in their respective prime locations)
  • Nitrogen may bond with up to four hydrogens in an organic compound to be called an amine. The most common amines are ammonia (NH3), and NH2)
  • Alcohols (carbon-hydrogen chains with OH groups attached to the ends)

What makes an amino acid basic?

  • An amine functional group. (Note that amino acids with amides on the side chain do NOT produce a basic amino acid)

What makes an amino acid acidic?

How a Reversible Reaction Works

Most reactions encountered in chemistry are irreversible reactions (or reversible, but with very little product converting back into reactant). For example, if you burn a piece of wood using the combustion reaction, you never see the ash spontaneously make new wood, do you? Yet, some reactions do reverse. How does this work?

The answer has to do with the energy output of each reaction and that required for it to occur. In a reversible reaction, reacting molecules in a closed system collide with each other and use the energy to break chemical bonds and form new products. Enough energy is present in the system for the same process to occur with the products. Bonds are broken and new ones formed, that happen to result in the initial reactants.

Fun Fact

At one time, scientists believed all chemical reactions were irreversible reactions. In 1803, Berthollet proposed the idea of a reversible reaction after observing the formation of sodium carbonate crystals on the edge of a salt lake in Egypt. Berthollet believed excess salt in the lake pushed the formation of sodium carbonate, which could then react again to form sodium chloride and calcium carbonate:

Waage and Guldberg quantified Berthollet's observation with the law of mass action that they proposed in 1864.


While reverse transcriptases have functional roles in biological systems, they also serve as important tools for studying RNA populations. One of the first molecular biology protocols utilizing reverse transcriptases was for the production of cDNA to build libraries that contained DNA copies of mRNA from cells and tissues [9,10]. These cDNA libraries aid in understanding actively expressed genes and their functions at a specific time point.

Although the creation of cDNA libraries was an important step forward in characterizing expressed genes, challenges remained for the study of low-abundance RNAs. These were subsequently addressed with the development of the polymerase chain reaction (PCR), a technique to amplify small amounts of genetic material. Reverse transcription combined with PCR, or reverse transcription PCR (RT-PCR), allows detection of RNA even at very low levels of gene expression and paves the way for detection of circulating RNA, RNA viruses, and cancerous gene fusions in molecular diagnostics [11-13].

In addition, cDNAs serve as templates in applications such as microarray and RNA sequencing to characterize unknown RNAs in a high-throughput manner [14-17]. (Learn more about reverse transcription applications.)

Principle of RT-PCR

Reverse transcription and PCR amplification can be performed as a two-step process in a single tube or with two separate reactions. In both cases, RNA is first reverse-transcribed into cDNA, which is then used as the template for PCR amplification.

  • Non-sequence-specific primers:
    • Random hexamers are a mixture of all possible combinations of six nucleotide sequences that can attach randomly to mRNA and initiate reverse transcription of the entire RNA pool.
    • Oligo-dT primers are complementary to the poly-A tail of mRNA molecules and allow synthesis of cDNA only from mRNA molecules.
    • Sequence-specific primers are the most restricted because they are designed to bind selectively to mRNA molecules of interest, which makes reverse transcription a target-specific process.

    One-step RT-PCR

    cDNA synthesis and PCR are performed in a single reaction vessel in a common reaction buffer. Gene-specific primers direct cDNA synthesis and amplification of a specific target. Major advantages of one-step reaction include minimal sample handling, reduced bench time, and closed-tube reactions, reducing chances for pipetting errors and cross-contamination.

    The quality and scarcity of RNA samples impact the efficiency of one-step RT-PCR. cDNA synthesis product cannot be saved after one-step RT-PCR so additional aliquots of the original RNA sample(s) are required in order to repeat reactions or to assess the expression of other genes.

    Two-step RT-PCR

    In two-step RT-PCR, cDNA synthesis is carried out using random hexamers, oligo-dT primers, and/or gene-specific primers which gives a mixture of cDNA molecules. cDNAs thus synthesized are amplified using specific primers.

    In two-step RT-PCR, cDNA is synthesized in one reaction, and an aliquot of the cDNA is then used for a subsequent PCR experiment. This requires extra open-tube step, more pipetting manipulations, and longer hands-on time which may lead to greater variability and risk of contamination. Remaining cDNA can be stored for future use, or quantitating the expression of multiple genes from a single RNA/cDNA sample.

    Applications of RT-PCR

    1. Dengue virus
    2. Hantavirus
    3. Human metapneumovirus
    4. Severe acute respiratory syndrome (SARS)

    Quantitative RT-PCR assays are commonly used for the detection of HIV and HCV viral load (amount of these viruses present in the blood of a patient) testing.

    Viral load data are important for monitoring the response of the individual patient to therapy. For instance, after appropriate antiretroviral therapy, patient infected with HIV virus should demonstrate an increase in CD4 count and a decrease in HIV viral load.

    RT-PCR may also be used to detect other microorganisms (bacteria, parasites, and fungi) by targeting their rRNA. This approach is better than detection of DNA, as the presence of RNA is more likely associated with the presence of viable organisms.

    Detection of mRNA using RT-PCR helps to study the gene expression of both microorganisms and human host cells.

    What is a catalyst?

    If you have a lot of work that needs to get done, doing it alone will take a significant amount of time. However, if someone helps you do the work, it speeds up the process and the work gets done at a faster rate. A catalyst does the same thing as an extra person it makes a process or chemical reaction begin or finish faster. An enzyme, which is also a catalyst, makes the human body&rsquos work easier by making the minute processes in the body occur faster.

    For example, a routine peptide bond break reaction would take approximately 400 years at room temperature, so we clearly need something to speed up the process. Enzymes speed up the reaction so dramatically, they might as well be our biological superheroes.

    The Flash, a character from DC Comics, can surpass the speed of light. (photo Credit : Pixabay)

    That example should give you some idea about the extent to which enzymes are required for bodily processes and sustainability. Given that enzymes are so significant for our metabolism, let&rsquos explore the details of their functioning and purpose.


    Enzymes speed the rate of chemical reactions. A catalyst is a chemical involved in, but not consumed in, a chemical reaction. Enzymes are proteins that catalyze biochemical reactions by lowering the activation energy necessary to break the chemical bonds in reactants and form new chemical bonds in the products. Catalysts bring reactants closer together in the appropriate orientation and weaken bonds, increasing the reaction rate. Without enzymes, chemical reactions would occur too slowly to sustain life.

    The functionality of an enzyme is determined by the shape of the enzyme. The area in which bonds of the reactant(s) are broken is known as the active site. The reactants of enzyme catalyzed reactions are called substrates. The active site of an enzyme recognizes, confines, and orients the substrate in a particular direction.

    Enzymes are substrate specific, meaning that they catalyze only specific reactions. For example, proteases (enzymes that break peptide bonds in proteins) will not work on starch (which is broken down by the enzyme amylase). Notice that both of these enzymes end in the suffix -ase. This suffix indicates that a molecule is an enzyme.

    Environmental factors may affect the ability of enzymes to function. You will design a set of experiments to examine the effects of temperature, pH, and substrate concentration on the ability of enzymes to catalyze chemical reactions. In particular, you will be examining the effects of these environmental factors on the ability of catalase to convert H2O2 into H2O and O2.

    Allosteric Inhibition (With Diagram) | Enzymes

    Sometimes it has been found that when a series of reactions is catalysed by a number of enzymes in sequence, the accumulation of the final end-product may cause inhibition in the activity of the first enzyme of the series. This inhibition due to a compound (final end product) which is totally different in structure from the substrate of the enzyme is called as allosteric in­hibition or feedback inhibition and such an enzyme is called as allosteric enzyme.

    This type of inhibition takes place due to the presence of allosteric site (Greek allo = ‘other’ stereos = ‘space’ or ‘site’) on the surface of the allosteric enzyme away from the active site. The final end-product molecule fits in the allosteric site and in some way brings about a change in shape of the enzyme so that the active site of the enzyme becomes unfit for making complex with its substrate. The allosteric inhibition is reversible. When the concentration of the final end product in the cell falls, it leaves the allosteric site, and the activity of the allosteric enzyme is re­stored.

    Allosteric inhibition is shown diagrammatically in Fig. 10.11.

    One of the classical and first discovered examples of allosteric inhibition is furnished by the bacterial enzyme system of E. coli which catalyses the conversion of L-Threonine into L-Isoleucine involving 5 different enzymes in sequence viz., 1. Threonine dehydratase 2. Acetolactate synthase 3. Ketoacid reductoisomerase 4. Dihydroxy acid dehydratase and 5. Transaminase (See Fig. 10.12).

    In this sequence, only the first enzyme i.e., threonine dehydratase is inhibited by Isoleucine which is the end-product of this sequence. The activity of this enzyme is neither inhibited by any other intermediate of the sequence, nor is any other enzyme of this sequence inhibited by Isoleucine.

    The inhibition of the first enzyme threonie dehydratase is reversible. When the concen­tration of isoleucine in the cells increases the activity of this enzyme is decreased so that production of isoleucine falls. But, when isoleucine concentration decreases, the activity of threo­nine dehydratase increases and the production of isoleucine in the cells is restored’.

    How Cells Work

    There are all sorts of enzymes at work inside of bacteria and human cells, and many of them are incredibly interesting! Cells use enzymes internally to grow, reproduce and create energy, and they often excrete enzymes outside their cell walls as well. For example, E. coli bacteria excrete enzymes to help break down food molecules so they can pass through the cell wall into the cell. Some of the enzymes you may have heard of include:

    • Proteases and peptidases - A protease is any enzyme that can break down a long protein into smaller chains called peptides (a peptide is simply a short amino acid chain). Peptidases break peptides down into individual amino acids. Proteases and peptidases are often found in laundry detergents -- they help remove things like blood stains from cloth by breaking down the proteins. Some proteases are extremely specialized, while others break down just about any chain of amino acids. (You may have heard of protease inhibitors used in drugs that fight the AIDS virus. The AIDS virus uses very specialized proteases during part of its reproductive cycle, and protease inhibitors try to block them to shut down the reproduction of the virus.)
    • Amylases - Amylases break down starch chains into smaller sugar molecules. Your saliva contains amylase and so does your small intestine. Maltase, lactase, sucrase (described in the previous section) finish breaking the simple sugars down into individual glucose molecules.
    • Lipases - Lipases break down fats.
    • Cellulases - Cellulases break cellulose molecules down into simpler sugars. Bacteria in the guts of cows and termites excrete cellulases, and this is how cows and termites are able to eat things like grass and wood.

    Bacteria excrete these enzymes outside their cell walls. Molecules in the environment are broken down into pieces (proteins into amino acids, starches into simple sugars, etc.) so they are small enough to pass through the cell's wall into the cytoplasm. This is how an E. coli eats!

    Inside a cell, hundreds of highly specialized enzymes carry out extremely specific tasks that the cell needs to live its life. Some of the more amazing enzymes found inside cells include:

    • Energy enzymes - A set of 10 enzymes allows a cell to perform glycolysis. Another eight enzymes control the citric-acid cycle (also known as the Krebs cycle). These two processes together allow a cell to turn glucose and oxygen into adenosine triphosphate, or ATP. In an oxygen-consuming cell like E. coli or a human cell, one glucose molecule forms 36 ATP molecules (in something like a yeast cell, which lives its life without oxygen, only glycosis occurs and it produces only two ATP molecules per glucose molecule). ATP is a fuel molecule that is able to power enzymes by performing "uphill" chemical reactions.
    • Restriction enzymes - Many bacteria are able to produce restriction enzymes, which recognize very specific patterns in DNA chains and break the DNA at those patterns. When a virus injects its DNA into a bacterium, the restriction enzyme recognizes the viral DNA and cuts it, effectively destroying the virus before it can reproduce.
    • DNA-manipulation enzymes - There are specialized enzymes that move along DNA strands and repair them. There are other enzymes that can untwist DNA strands to reproduce them (DNA polymerase). Still others can find small patterns on DNA and attach to them, blocking access to that section of DNA (DNA-binding proteins).
    • Enzyme-production enzymes - All of these enzymes have to come from somewhere, so there are enzymes that produce the cell's enzymes! Ribonucleic acid (RNA), in three different forms (messenger RNA, transfer RNA and ribosomal RNA), is a big part of the process.

    A cell really is nothing but a set of chemical reactions, and enzymes make those reactions happen properly.