1. Enzymes are biological catalysts that speed up chemical reactions without being consumed in the process. They do this by lowering the activation energy of reactions.
2. Most enzymes are proteins that contain an active site where substrates bind and reactions occur. Enzymes exhibit high specificity and can accelerate reactions by factors of millions.
3. The Michaelis-Menten model describes enzyme kinetics, showing that reaction velocity is determined by the concentration of the enzyme-substrate complex and reaches a maximum velocity (Vmax) as substrate concentration increases. This model is important for understanding how enzymes function.
What is enzyme?
How enzyme catalyze the reaction
Enzyme kinetics
History
Enzyme kinetic equation
Michaelis-menten equation
Michaelis-menten curve
Michaelis-menten equation derivation
Reversible inhibition
Two substrate reaction
Conclusion
References
A comprehensive coverage of Enzymes including basics, mechanisms of enzyme catalysis, enzyme inhibition and clinical applications, mostly based on Stryer- Biochemistry. The slides were intended for MBBS teaching, but should benefit the students of Biochemistry and allied sciences.
Prepared in Sept 2015
This ppt includes overall idea of what is enzymes, how it works, mechanism of enzymes, kinetics and how to inhibit enzyme activities. The reference is the ideal book for biochemistry - Lehninger . Understanding is easy for everyone.
Enzyme Kinetics and thermodynamic analysisKAUSHAL SAHU
Â
Introduction
Kinetics and thermodynamicSG
Thermodynamic in enzymatic reactions
balanced equations in chemical reactions
changes in free energy determine the direction & equilibrium state of chemical reactions
the rates of reactions
Factors effecting enzymatic activity
(i) Enzyme concentration.
(ii) Substrate concentration.
(iii)Temperature
(iv) pH.
(v) Activators.
(vi)Inhibitors
Michaelis-menten equation
CONCLUSIONS
REFERENECES
What is enzyme?
How enzyme catalyze the reaction
Enzyme kinetics
History
Enzyme kinetic equation
Michaelis-menten equation
Michaelis-menten curve
Michaelis-menten equation derivation
Reversible inhibition
Two substrate reaction
Conclusion
References
A comprehensive coverage of Enzymes including basics, mechanisms of enzyme catalysis, enzyme inhibition and clinical applications, mostly based on Stryer- Biochemistry. The slides were intended for MBBS teaching, but should benefit the students of Biochemistry and allied sciences.
Prepared in Sept 2015
This ppt includes overall idea of what is enzymes, how it works, mechanism of enzymes, kinetics and how to inhibit enzyme activities. The reference is the ideal book for biochemistry - Lehninger . Understanding is easy for everyone.
Enzyme Kinetics and thermodynamic analysisKAUSHAL SAHU
Â
Introduction
Kinetics and thermodynamicSG
Thermodynamic in enzymatic reactions
balanced equations in chemical reactions
changes in free energy determine the direction & equilibrium state of chemical reactions
the rates of reactions
Factors effecting enzymatic activity
(i) Enzyme concentration.
(ii) Substrate concentration.
(iii)Temperature
(iv) pH.
(v) Activators.
(vi)Inhibitors
Michaelis-menten equation
CONCLUSIONS
REFERENECES
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Global launch of the Healthy Ageing and Prevention Index 2nd wave â alongside...ILC- UK
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The Healthy Ageing and Prevention Index is an online tool created by ILC that ranks countries on six metrics including, life span, health span, work span, income, environmental performance, and happiness. The Index helps us understand how well countries have adapted to longevity and inform decision makers on what must be done to maximise the economic benefits that comes with living well for longer.
Alongside the 77th World Health Assembly in Geneva on 28 May 2024, we launched the second version of our Index, allowing us to track progress and give new insights into what needs to be done to keep populations healthier for longer.
The speakers included:
Professor Orazio Schillaci, Minister of Health, Italy
Dr Hans Groth, Chairman of the Board, World Demographic & Ageing Forum
Professor Ilona Kickbusch, Founder and Chair, Global Health Centre, Geneva Graduate Institute and co-chair, World Health Summit Council
Dr Natasha Azzopardi Muscat, Director, Country Health Policies and Systems Division, World Health Organisation EURO
Dr Marta Lomazzi, Executive Manager, World Federation of Public Health Associations
Dr Shyam Bishen, Head, Centre for Health and Healthcare and Member of the Executive Committee, World Economic Forum
Dr Karin Tegmark Wisell, Director General, Public Health Agency of Sweden
QA Paediatric dentistry department, Hospital Melaka 2020Azreen Aj
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QA study - To improve the 6th monthly recall rate post-comprehensive dental treatment under general anaesthesia in paediatric dentistry department, Hospital Melaka
Leading the Way in Nephrology: Dr. David Greene's Work with Stem Cells for Ki...Dr. David Greene Arizona
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As we watch Dr. Greene's continued efforts and research in Arizona, it's clear that stem cell therapy holds a promising key to unlocking new doors in the treatment of kidney disease. With each study and trial, we step closer to a world where kidney disease is no longer a life sentence but a treatable condition, thanks to pioneers like Dr. David Greene.
Telehealth Psychology Building Trust with Clients.pptxThe Harvest Clinic
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Telehealth psychology is a digital approach that offers psychological services and mental health care to clients remotely, using technologies like video conferencing, phone calls, text messaging, and mobile apps for communication.
Antibiotic Stewardship by Anushri Srivastava.pptxAnushriSrivastav
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Stewardship is the act of taking good care of something.
Antimicrobial stewardship is a coordinated program that promotes the appropriate use of antimicrobials (including antibiotics), improves patient outcomes, reduces microbial resistance, and decreases the spread of infections caused by multidrug-resistant organisms.Â
WHO launched the Global Antimicrobial Resistance and Use Surveillance System (GLASS) in 2015 to fill knowledge gaps and inform strategies at all levels.
ACCORDING TO apic.org,
Antimicrobial stewardship is a coordinated program that promotes the appropriate use of antimicrobials (including antibiotics), improves patient outcomes, reduces microbial resistance, and decreases the spread of infections caused by multidrug-resistant organisms.
ACCORDING TO pewtrusts.org,
Antibiotic stewardship refers to efforts in doctorsâ offices, hospitals, long term care facilities, and other health care settings to ensure that antibiotics are used only when necessary and appropriate
According to WHO,
Antimicrobial stewardship is a systematic approach to educate and support health care professionals to follow evidence-based guidelines for prescribing and administering antimicrobials
In 1996, John McGowan and Dale Gerding first applied the term antimicrobial stewardship, where they suggested a causal association between antimicrobial agent use and resistance. They also focused on the urgency of large-scale controlled trials of antimicrobial-use regulation employing sophisticated epidemiologic methods, molecular typing, and precise resistance mechanism analysis.
 Antimicrobial Stewardship(AMS) refers to the optimal selection, dosing, and duration of antimicrobial treatment resulting in the best clinical outcome with minimal side effects to the patients and minimal impact on subsequent resistance.
According to the 2019 report, in the US, more than 2.8 million antibiotic-resistant infections occur each year, and more than 35000 people die. In addition to this, it also mentioned that 223,900 cases of Clostridoides difficile occurred in 2017, of which 12800 people died. The report did not include viruses or parasites
VISION
Being proactive
Supporting optimal animal and human health
Exploring ways to reduce overall use of antimicrobials
Using the drugs that prevent and treat disease by killing microscopic organisms in a responsible way
GOAL
to prevent the generation and spread of antimicrobial resistance (AMR). Doing so will preserve the effectiveness of these drugs in animals and humans for years to come.
being to preserve human and animal health and the effectiveness of antimicrobial medications.
to implement a multidisciplinary approach in assembling a stewardship team to include an infectious disease physician, a clinical pharmacist with infectious diseases training, infection preventionist, and a close collaboration with the staff in the clinical microbiology laboratoryÂ
 to prevent antimicrobial overuse, misuse and abuse.
to minimize the developme
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Defecation
Normal defecation begins with movement in the left colon, moving stool toward the anus. When stool reaches the rectum, the distention causes relaxation of the internal sphincter and an awareness of the need to defecate. At the time of defecation, the external sphincter relaxes, and abdominal muscles contract, increasing intrarectal pressure and forcing the stool out
The Valsalva maneuver exerts pressure to expel faeces through a voluntary contraction of the abdominal muscles while maintaining forced expiration against a closed airway. Patients with cardiovascular disease, glaucoma, increased intracranial pressure, or a new surgical wound are at greater risk for cardiac dysrhythmias and elevated blood pressure with the Valsalva maneuver and need to avoid straining to pass the stool.
Normal defecation is painless, resulting in passage of soft, formed stool
CONSTIPATION
Constipation is a symptom, not a disease. Improper diet, reduced fluid intake, lack of exercise, and certain medications can cause constipation. For example, patients receiving opiates for pain after surgery often require a stool softener or laxative to prevent constipation. The signs of constipation include infrequent bowel movements (less than every 3 days), difficulty passing stools, excessive straining, inability to defecate at will, and hard feaces
IMPACTION
Fecal impaction results from unrelieved constipation. It is a collection of hardened feces wedged in the rectum that a person cannot expel. In cases of severe impaction the mass extends up into the sigmoid colon.
DIARRHEA
Diarrhea is an increase in the number of stools and the passage of liquid, unformed feces. It is associated with disorders affecting digestion, absorption, and secretion in the GI tract. Intestinal contents pass through the small and large intestine too quickly to allow for the usual absorption of fluid and nutrients. Irritation within the colon results in increased mucus secretion. As a result, feces become watery, and the patient is unable to control the urge to defecate. Normally an anal bag is safe and effective in long-term treatment of patients with fecal incontinence at home, in hospice, or in the hospital. Fecal incontinence is expensive and a potentially dangerous condition in terms of contamination and risk of skin ulceration
HEMORRHOIDS
Hemorrhoids are dilated, engorged veins in the lining of the rectum. They are either external or internal.
FLATULENCE
As gas accumulates in the lumen of the intestines, the bowel wall stretches and distends (flatulence). It is a common cause of abdominal fullness, pain, and cramping. Normally intestinal gas escapes through the mouth (belching) or the anus (passing of flatus)
FECAL INCONTINENCE
Fecal incontinence is the inability to control passage of feces and gas from the anus. Incontinence harms a patientâs body image
PREPARATION AND GIVING OF LAXATIVESACCORDING TO POTTER AND PERRY,
An enema is the instillation of a solution into the rectum and sig
Medical Technology Tackles New Health Care Demand - Research Report - March 2...pchutichetpong
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M Capital Group (âMCGâ) predicts that with, against, despite, and even without the global pandemic, the medical technology (MedTech) industry shows signs of continuous healthy growth, driven by smaller, faster, and cheaper devices, growing demand for home-based applications, technological innovation, strategic acquisitions, investments, and SPAC listings. MCG predicts that this should reflects itself in annual growth of over 6%, well beyond 2028.
According to Chris Mouchabhani, Managing Partner at M Capital Group, âDespite all economic scenarios that one may consider, beyond overall economic shocks, medical technology should remain one of the most promising and robust sectors over the short to medium term and well beyond 2028.â
There is a movement towards home-based care for the elderly, next generation scanning and MRI devices, wearable technology, artificial intelligence incorporation, and online connectivity. Experts also see a focus on predictive, preventive, personalized, participatory, and precision medicine, with rising levels of integration of home care and technological innovation.
The average cost of treatment has been rising across the board, creating additional financial burdens to governments, healthcare providers and insurance companies. According to MCG, cost-per-inpatient-stay in the United States alone rose on average annually by over 13% between 2014 to 2021, leading MedTech to focus research efforts on optimized medical equipment at lower price points, whilst emphasizing portability and ease of use. Namely, 46% of the 1,008 medical technology companies in the 2021 MedTech Innovator (âMTIâ) database are focusing on prevention, wellness, detection, or diagnosis, signaling a clear push for preventive care to also tackle costs.
In addition, there has also been a lasting impact on consumer and medical demand for home care, supported by the pandemic. Lockdowns, closure of care facilities, and healthcare systems subjected to capacity pressure, accelerated demand away from traditional inpatient care. Now, outpatient care solutions are driving industry production, with nearly 70% of recent diagnostics start-up companies producing products in areas such as ambulatory clinics, at-home care, and self-administered diagnostics.
The dimensions of healthcare quality refer to various attributes or aspects that define the standard of healthcare services. These dimensions are used to evaluate, measure, and improve the quality of care provided to patients. A comprehensive understanding of these dimensions ensures that healthcare systems can address various aspects of patient care effectively and holistically. Dimensions of Healthcare Quality and Performance of care include the following; Appropriateness, Availability, Competence, Continuity, Effectiveness, Efficiency, Efficacy, Prevention, Respect and Care, Safety as well as Timeliness.
2. Catalyst
⢠substance that increase rates of a
chemical reaction
⢠does not effect equilibrium
⢠remain unchanged in overall
process
⢠reactants bind to catalyst,
products are released
2
3. 3
⢠Enzymes are biological catalysts.
⢠Recall that by definition, catalysts alter the
rates of chemical reactions but are neither
formed nor consumed during the reactions
they catalyze.
⢠Enzymes are the most sophisticated
catalysts known.
⢠Most enzymes are proteins. Some nucleic
acids exhibit enzymatic activities (e.g.,
rRNA). We will focus primarily on protein-
type catalysts.
4. Catalysts increase product formation by
(1) lowering the energy barrier (activation energy)
for the product to form
(2) increases the favorable orientation of
colliding reactant molecules for product
formation to be successful (stabilize transition
state intermediate)
4
5. 5
Thermodynamics governs enzyme reactions, just
the same as with other chemical reactions.
Gibbâs âFree Energy,â ÎG, determines the
spontaneity of a reaction:
⢠ÎG must be negative for a reaction to occur
spontaneously (âexergonicâ).
⢠A system is at equilibrium and no net change can
occur if ÎG is zero.
⢠A reaction will not occur spontaneously if ÎG is
positive (âendergonicâ); to proceed, it must
receive an input of free energy from another
source.
6. For the reaction: A + B â C + D,
ÎG = ÎGo + RT ln [C][D]
[A][B]
ÎG = ÎGo + RT ln Keq
⢠At 25°C, when Keq changes by 10-fold, ÎG
changes by only 1.36!
⢠Small changes in ÎG describe HUGE
changes in Keq.
Note: ÎGoâ or ÎGâ denotes pH=7
8. 8
Enzymes â Activation Energy
Uncatalyzed Reaction: Catalyzed Reaction:
Lower activation energy (ÎGâĄ) increases the rate of reaction,
reaching equilibrium faster.
In this case, ÎG remains unchanged. Thus, the final ratio of
products to reactants at equilibrium is the same in both cases.
ÎG
âĄ
ÎG
âĄ
Î
G
Î
G
10. 10
⢠In biochemistry, we use slightly
different terms for the participants in a
reaction:
Traditional Biochemistry
Reactant Substrate
Catalyst Enzyme
Product Product
11. Catalytic Power
⢠Enzymes can accelerate reactions
as much as 1016 over uncatalyzed
rates!
⢠Urease is a good example:
â Catalyzed rate: 3x104/sec
â Uncatalyzed rate: 3x10 -10/sec
â Ratio is 1x1014 !
11
12. Specificity
⢠Enzymes selectively recognize
proper substrates over other
molecules
⢠Enzymes produce products in very
high yields - often much greater
than 95%
⢠Specificity is controlled by
structure - the unique fit of
substrate with enzyme controls the
selectivity for substrate and the
product yield 12
13. Classes of enzymes
1. Oxidoreductases = catalyze oxidation-
reduction reactions (NADH)
2. Transferases = catalyze transfer of functional
groups from one molecule to another.
3. Hydrolases = catalyze hydrolytic cleavage
4. Lyases = catalyze removal of a group from or
addition of a group to a double bond, or other
cleavages involving electron rearrangement.
5. Isomerases = catalyze intramolecular
rearrangement.
6. Ligases = catalyze reactions in which two
molecules are joined.
Enzymes named for the substrates and type of
reaction
13
15. PROSTHETIC GROUPS
⢠Many enzymes contain small nonprotein
molecules and metal ions that participate
directly in substrate binding or catalysis.
Termed prosthetic groups, cofactors, and
coenzymes.
⢠Prosthetic groups are distinguished by their
tight, stable incorporation into a proteinâs
structure by covalent or noncovalent forces e.g.
pyridoxal phosphate, flavin mononucleotide
(FMN), flavin dinucleotide (FAD), thiamin
pyrophosphate, biotin, and the metal ions of Co,
Cu, Mg, Mn, Se, and Zn (metalloenzymes).
15
16. COFACTORS
⢠They bind in a transient, dissociable manner
either to the enzyme or to a substrate such as
ATP.
⢠Cofactors must be present in the medium
surrounding the enzyme for catalysis to occur.
⢠The most common cofactors also are metal ions.
⢠Enzymes that require a metal ion cofactor are
termed METAL-ACTIVATED ENZYMES to
distinguish them from the METALLOENZYMES
for which metal ions serve as prosthetic groups.
16
17. COENZYMES
⢠They serve as recyclable shuttlesâor group
transfer reagentsâthat transport many
substrates from their point of generation to
their point of utilization.
⢠Association with the coenzyme also stabilizes
substrates such as hydrogen atoms or hydride
ions.
⢠Other substance transported are methyl groups
(folates), acyl groups (coenzyme A), and
oligosaccharides (dolichol) â thiamin, riboflavin,
niacin, biotin
⢠Enzyme + Co-enzyme = holoenzyme
⢠Enzyme alone = apoenzyme 17
18. 18
⢠For enzymes to function, they must come in
contact with the substrate.
⢠While in contact, they are referred to as
the âenzyme-substrate complex.â
⢠The high specificity of many enzymes led to
the hypothesis that enzymes were similar to
a lock⌠and the substrate was like a key:
(Fischer, 1890)
⢠In 1958, Koshland proposed that the enzyme
changes shape to fit the incoming substrate.
This is called an âinduced fit.â
20. 20
⢠Enzymes are often quite large compared to
their substrates. The relatively small region
where the substrate binds and catalysis takes
place is called the âactive site.â (e.g., human
carbonic anhydrase:)
21. 21
⢠General Characteristics of Active Sites:
â The active site takes up a relatively small
part of the total volume of an enzyme
â The active site is a 3-dimensional
â cleft or crevice.
â Water is usually excluded unless it is a
reactant.
â Substrates bind to enzymes by multiple
weak attractions (electrostatic interactions,
hydrogen bonds, hydrophobic interactions,
etc.
â Specificity of binding depends on precise
spatial arrangement of atoms in space.
22. Kinetics
⢠study of reaction rate
⢠determines number of steps involved
⢠determines mechanism of reaction
⢠identifies ârate-limitingâ step
22
23. 23
⢠In 1913, two women scientists, Leonor
Michaelis and Maud Menten proposed a simple
model to account for the kinetic
characteristics of enzymes*.
Leonor
Michaelis?
Dr. Maud Menten
24. 24
What was Michaelisâ and Mentonâs contribution?
Since the enzyme and substrate must form the ES complex
before a reaction can take place, they proposed that the rate
of the reaction depended upon the concentration of ES:
E + S ES E + P
k1
k-1
k2
k-2
They also proposed that at the beginning of the reaction, very
little product returned to form ES. Therefore, k-2 was
extremely small and could be ignored:
E + S ES E + P
k1
k-1
k2
26. 26
E + S ES E + P
k1
k2
k3
The rate (Velocity) of the appearance of product, depends on [ES]:
V = k3[ES]
ES has two fates:
1. Go to product
2. Reverse back enzyme + substrate
When the catalyzed reaction is running smoothly and producing product
at a constant rate, the concentration of ES is constant at we say that
the reaction has reached a âsteady state.â Therefore, we may say
that the rates for formation of ES and the breakdown of ES are
equal:
Rate of ES Formation d[ES]/dt = k1[E][S]
Rate of ES Breakdown -d[ES]/dt = k2[ES] + k3[ES]
At the âsteady state:â d[ES]/dt = 0 = k1[E][S] â (k2+k3)[[ES]
Rearranging: k1[E][S] = (k2+k3)[[ES]
27. 27
Steady State: k1[E][S] = (k2+k3)[[ES]
Rearrange, solving for [ES]: [ES] = [E][S] k 1 .
k2 + k3
Define M&M constant: Km: .. Km = k2 + k3 .
(âDissociationâ) k1
Result: [ES] = [E][S] / Km
If: [E] <<<[S], then [S] â [ES] â [S]
Since: [Et] = [E] + [ES], it follows that [E] = [Et] â [ES]
Substituting for [E]: [ES] = ([Et] â [ES]) [S] / Km
Solving for [ES]: [ES] = [Et][S] / Km .
1+ [S] / Km
Simplifying: [Es] = [Et] [S]
[S] + Km
28. 28
Steady State: k1[E][S] = (k2+k3)[[ES]
Rearrange, solving for [ES]: [ES] = [E][S] k 1 .
k2 + k3
Define M&M constant: Km:. Km = k2 + k3 .
k1
Result: [ES] = [E][S] / Km
If: [E] <<<[S], then [S] â [ES] â [S]
Since: [Et] = [E] + [ES], it follows that [E] = [Et] â [ES]
Substituting for [E]: [ES] = ([Et] â [ES]) [S] / Km
Solving for [ES]:* [ES] = [Et][S] / Km .
1+ [S] / Km
Simplifying:* [Es] = [Et] [S]
[S] + Km
*Class Assignment: Show this algebreic rearrangement. Submit during next lecture period.
29. 29
Now that we have an expression V = k3 [ES]
for [ES], we substitute into our V = k3 [Et] [S] .
âvelocityâ equation: [S] + Km
Consider [S] and Km: V = k3 [Et] [S] .
[S]+Km
As [S] â â, then [S] â 1
[S]+Km
We can define maximal velocity Vmax = k3 [Et]
as the velocity when [S] = â.
(We also assume that under these conditions, all enzymes [Et] are bound to S in the ES complex. )
The rate constant, k3, is the âturnover number,â or the maximum number of
substrates can be converted to products by a single enzyme molecule.
Therefore: V = Vmax [S]
(M&M Equation) [S] + Km
30. 30
(M&M Equation) V = Vmax [S]
[S] + Km
What does this equation describe?
⢠It describes the velocity of an enzyme-catalyzed reaction at different
concentrations of substrate [S].
⢠It helps determine the maximum velocity of the catalyzed reaction.
⢠It assigns a value for Km, the âMichaelis constant,â that is inversely
proportional to the affinity of the enzyme for its substrate.
How is this equation utilized in the laboratory?
⢠A series of test tubes are prepared, all with identical concentrations of
enzyme, but increasing concentrations of substrate.
⢠The velocity of each tube increases as the substrate increases.
⢠A plot of the results is hyperboic, reaching an asymptote we define as
Vmax.
31. 31
Why does the velocity reach a maximum?
V = Vmax [S]
[S] + Km
32. 32
The Michaelis-Menton
equation was a pivotal
contribution to
understanding how
enzymes functioned.
However, during routine
use in the laboratory, it
was difficult to estimate
Vmax. Everyone had
different ideas the
actual value for Vmax.
Since it is impossible to
actually make a solution
with infinite
concentration of
substrate, a different
equation was needed.
33. 33
A relatively simple solution was provided by Lineweaver and Burke, who simply suggested
that the M&M equation be inverted. This would yield a âdouble inverse plotâ that is
linear:
(M&M Equation) V = Vmax [S]
[S] + Km
Inverting the Equation yields: 1 = Km 1 + 1 .
(Lineweaver-Burke Equation) V Vmax [S] Vmax
By plotting 1/ V as a function of 1/[S],
a linear plot is obtained:
Slope = Km/Vmax
y-intercept = 1/Vmax
34. 34
Comparision of these two methods of plotting the same data:
Michaelis-Menton Equation: Linewaver-Burke Equation:
36. 36
Factors Affecting Activity
Temperature affects enzyme activity. Higher
temperatures mean molecules are moving
faster and colliding more frequently.
Up to a certain point, increases in temperature
increase the rates of enzymatic reactions.
Excess heat can denature the enzyme, causing
a permanent loss of activity.
Examples:
⢠Cooking denatures many enzymes, killing
bacteria and inactivating viruses, parasites,
etc.
⢠Grass grows faster during the hot summer
than during the cooler spring or fall.
⢠Insects cannot move as fast in cold
weather as they can on a hot day.
⢠Operating rooms are often cooled down to
slow a patientâs metabolism during surgery.
37. 37
pH often affects enzymatic reaction rates. The âoptimum pHâ refers to the pH
at which the enzyme exhibits maximum activity. This pH varies from enzyme
to enzyme:
38. Km = [S] @ ½ Vmax
(units moles/L=M)
(1/2 of enzyme bound to S)
Vmax = velocity where all of the
enzyme is bound to substrate
(enzyme is saturated with S)
38
39. What does Km mean?
1. Km = [S] at ½ Vmax
2. Km is a combination of rate constants
describing the formation and breakdown of
the ES complex
3. Km is usually a little higher than the
physiological [S]
39
40. Limitations of M-M
1. Some enzyme catalyzed rxns show more complex behavior
E + S<->ES<->EZ<->EP<-> E + P
With M-M can look only at rate limiting step
2. Often more than one substrate
E+S1<->ES1+S2<->ES1S2<->EP1P2<-> EP2+P1<-> E+P2
Must optimize one substrate then calculate kinetic
parameters for the other
3. Assumes k-2 = 0
4. Assume steady state conditions
40
41. Enzyme Inhibition
⢠Inhibitor â substance that binds to an enzyme and interferes
with its activity
⢠Can prevent formation of ES complex or prevent ES
breakdown to E + P.
⢠Irreversible and Reversible Inhibitors
⢠Irreversible inhibitor binds to enzyme through covalent
bonds (binds irreversibly)
⢠Reversible Inhibitors bind through non-covalent interactions
(disassociates from enzyme)
⢠Why important?
41
42. Enzyme Inhibitor Types
⢠Inhibitors of enzymes are generally
molecules which resemble or mimic a
particular enzymes substrate(s). Therefore,
it is not surprising that many therapeutic
drugs are some type of enzyme inhibitor.
The modes and types of inhibitors have been
classified by their kinetic activities and sites
of actions. These include Reversible
Competitive Inhibitors, Reversible Non-
Competitive Inhibitors, and Irreversible
Inhibitors
43. Reversible Inhibitors
E + S <-> ES -> E + P
E + I <-> EI
Ki = [E][I]/[EI]
⢠Competitive
⢠Uncompetitive
⢠Non-competitive
43
45. Competitive Inhibitor (CI)
â˘CI binds free enzyme
â˘Competes with substrate for enzyme binding.
â˘Raises Km without effecting Vmax
â˘Can relieve inhibition with more S
45
46. 46
The antibiotic sulfanilamide was first discovered in 1932. Sulfanilamides and its
derivatives are called âsulfa drugs.â
Sulfanilamide is structurally similar to p-aminobenzoic acid (PABA), that is
required by many bacteria to produce an important enzyme cofactor, folic acid.
Sulfanilamide acts as a competitive inhibitor to enzymes that convert PAGA
into folic acid, resulting in a depletion of this cofactor. This results in
retarded growth and eventual death of the bacteria. (Mammals absorb their
folic acid from their diets, so sulfanilamide exerts no effects on them.)
47. 47
By adding various functional groups to the basic structure,
increased effectiveness has been achieved:
48. 48
Methotrexate is a competetive inhibitor for the coenzyme tetrahydrofolate
(required for proper activity of the enzyme dihydrofolate reductase). This
enzyme assists in the biosynthesis of purines and pyrimidines.
Methotrexate binds 1,000-fold more tightly to this enzyme than tetrahydrofolate,
significantly reducing nucleotide base synthesis. It is used to treat cancer.
49. Uncompetitive Inhibitor (UI)
â˘UI binds ES complex
â˘Prevents ES from proceeding to E + P or back to E + S.
â˘Lowers Km & Vmax, but ratio of Km/Vmax remains the same
â˘Occurs with multisubstrate enzymes
49
50. Non-competitive Inhibitor (NI)
â˘NI can bind free E or ES complex
â˘Lowers Vmax, but Km remains the same
â˘NIâs donât bind to S binding site therefore donât effect Km
â˘Alters conformation of enzyme to effect catalysis but not
substrate binding 50
51. ⢠Irreversible inhibitors generally result in the destruction
or modification of an essential amino acid required for
enzyme activity.
â˘
⢠Frequently, this is due to some type of covalent link
between enzyme and inhibitor.
⢠These types of inhibitors range from fairly simple,
broadly reacting chemical modifying reagents (like
iodoacetamide that reacts with cysteines) to complex
inhibitors that interact specifically and irreversibly with
active site amino acids. (termed suicide inhibitors).
Irreversible Inhibitors
52. ⢠These inhibitors are designed to mimic the
natural substrate in recognition and binding
to an enzyme active site.
⢠Upon binding and some catalytic
modification, a highly reactive inhibitor
product is formed that binds irreversibly and
inactivates the enzyme.
⢠Use of suicide inhibitors have proven to be
very clinically effective
53. 53
Enzymes â Inhibition
Irreversible Inhibitors are toxic. In the laboratory they can be used to map the
active site. These inhibitors often form covalent linkages to amino acids at the
active site.
DIPF (diisopropylphosphofluoridate) forms a covalent linkage to serine. If serine
plays an important catalytic role for the enzyme, DIPF can permanantly disable
the enzyme. Acetycholinesterase is an excellent example of DIPF inactivation
(making agents such as DIPF potent nerve agents):
54. 54
Enzymes â Inhibition
Another example of irreversible inhibition by covalent modification
is the reaction between iodoacetamide and a critical cysteine
residue:
55. 55
Enzyme Inhibition â Penicillin
Penicillin is a classic irreversible enzyme inhibitor, acting on bacterial
âtranspeptidase.â This enzyme strengthens bacterial cells walls, by
forming peptide bonds between D-amino acids that cross link the
peptidoglycan structure in cell walls.
Penicillin contains a beta-lactam ring (cyclic amide) fused to a thiazolidine
ring:
56. 56
Enzyme Inhibition â Penicillin
Penicillinâs structure is VERY SIMILAR to the normal
substrate for this enzyme.
In fact, penicillin is drawn into the active site of the
transpeptidase enzyme much like a competetive
inhibitor would be, due to its structural similarity:
57. 57
Enzyme Inhibition â Penicillin
Upon binding to the active site, the beta-lactam ring
opens and forms a covalent linkage to a serine at the
active site, permanently deactivating the enzyme:
58. Biochemistry 3070 â Enzymes 58
Enzyme Inhibition â Penicillin
Over the years, organic
chemists altered the
basic penicillin molecule,
adding groups for
better acid resistance
and a broader
antibacterial activity
spectrum.
âPenVKâ is the trade name
for
âPenicillin V, potassium
salt.â
Due to the structural
similarities between
these âcillins,â allergies
to one type of cillin,
extend throughout the
entire group of âbeta-
lactams.â
60. Regulation of Enzyme Activity
Enzyme quantity â regulation of gene expression (Response time =
minutes to hours)
a) Transcription
b) Translation
c) Enzyme turnover
Enzyme activity (rapid response time = fraction of seconds)
a) Allosteric regulation
b) Covalent modification
c) Association-disassociationâ
d) Proteolytic cleavage of proenzyme
60
61. Allosteric Regulation
⢠End products are often inhibitors
⢠Allosteric modulators bind to site other
than the active site
⢠Allosteric enzymes usually have 4o
structure
⢠Vo vs [S] plots give sigmoidal curve for
at least one substrate
⢠Can remove allosteric site without
effecting enzymatic action
61