MLS2213 Unit 10

Enymes

The first enzyme to be measured for clinical purposes was , higher levels of which is associated with a greater _

LDH, tumor burden

Enzymes are this type of molecule

protein

Enzymes participate in but are not by a reaction

consumed

An alteration in the structure of an enzyme will also alter its

functionality

Enzymes may require one or both of these to catalyze a reaction

coenzyme, activator

Coenzymes are often ____ acceptors or donors

hydrogen

Activators are usually

metal cations

An enzyme composed of more than one protein subunit is said to have

quarternary structure

When subunits have minor variations leading to multiple forms of a functionally identical enzyme, it is said to be a(n)

isoenzyme

These two enzymes are particularly well known for having isoenzymes

CK, LDH

The two possible subunit types of CK are

M, B

The two possible subunit types of LDH are

L, H

Isoenzymes are often produced mostly by one organ or tissue type, allowing some degree of

specificity

Enzymes are found in the blood after

leaking out of cells

Although enzymes in the bloodstream are still functional, they typically

serve no purpose

In a healthy individual, the rate at which enzymes leak from cells is

known

Cellular stress or disease may cause enzymes to leak from cells due to

increased cell membrane permeability

Cellular necrosis may cause enzymes to escape cells due to

rupture

An overall increase in blood enzyme levels may occur when cells are

proliferating

Enzymes may be found in elevated levels in the bloodstream even if a normal percentage is leaking out of cells if

enzyme production is high

When enzyme levels are below the reference range, it is typically

clinically insignificant

Enzymes with very low specificity, like LDH, provide very good _ for presence of disease.

sensitivity

LDH is found in this metabolic pathway

ebden-meyerhoff

These two enzymes are assayed by mass quantitation

prostatic acid phosphotase, CK-MB

Most enzymes are indirectly measured based on their

activity

Enzyme activity is

rate at a reaction is catalyzed

In the context of enzyme quantitation, rate of reaction is defined as

absorbance change per minute

Absorbance change is due to the of substrate or the of product or coenzyme.

disappearance, appearance

A common target for enzyme assays is the appearance or disappearance of

NADH

This type of assay is not ideal for measuring enzyme activity

endpoint

This type of assay is most ideal for measuring enzyme activity

multipoint kinetic

At the beginning of a reaction, reaction rate is slow and this is termed

lag phase

After lag phase, reaction rate increases in a linear fashion until

enzymes are saturated

Once all enzymes are saturated, the reaction rate remains stable and the reaction is said to be in

equilibrium

Enzyme assays must use absorbance readings taken when the reaction is in the _ phase in order to be accurate

linear rate

This equation gives provides an enzyme activity result in

international units

In this equation, ε is the

molar absorptivity constant

One international unit is the amount of enzyme activity that will catalyze _ of substrate into product per ____.

one micromole, minute

In an enzyme assay, the only _ should be the amount of enzyme present.

rate limiting factor

Enzyme assays operate on the assumption that and are proportional.

reaction rate, amount of enzyme

Substrate concentration, coenzyme concentration, temperature, pH, and presence of inhibitors are all

rate limiting factors

Most commonly, inhibitors are

anticoagulants

The fastest a reaction can go with a given amount of enzyme and unlimited substrate is

vmax

The amount of substrate that will give you half of the maximum velocity is

Km

Km may be thought of as the amount of substrate at which _ of enzymes are saturated.

half

On this chart, A represents

vmax

On this chart, B represents

km

When substrate concentration is low, a 10% increase in substrate concentration will result in

10% increase in rate

When substrate concentration is high, a 10% increase in substrate concentration will result in

no effect on rate

For LDH assays, substrate level must be _x that of the Km to prevent substrate exhaustion

20

For most enzyme assays, substrate level must be _x that of the Km to prevent substrate exhaustion

100

Enzyme reagents are typically buffered to the _ of the enzyme in question

optimal pH

Enzyme assays are typically performed at ___ or ___℃, which is lower than the typical optimum temperature

30, 37

Below the point of denaturation, each 10℃ increase in temperature typically will

double the reaction rate

Each 1℃ increase in temperature increases reaction rate by

10%

pH and temperature affect enzymes by altering the

active site

A chemical which decreases the rate of a reaction is a(n)

inhibitor

This type of inhibitor can be overcome by simply adding more substrate

reversible

Reversible inhibitors typically are

competitive with substrate

This type of inhibitor cannot be overcome by simply adding more substrate

irreversible

A location on an enzyme where an inhibitor might bind that is NOT the active site is termed a(n)

allosteric site

This type of chart can be used to determine if an inhibitor is present

lineweaver-burk plot

The lineweaver-burk plot is also known as the _ grap

double reciprocal

On this Lineweaver-Burk plot, B represents

normal rate

On this Lineweaver-Burk plot, C represents

non-competitive inhibitor

If an enzyme reaction is measured when the reaction is not in zero order, rates will appear falsely

low

In the presence of extremely high levels of , the reaction rate may be so fast that measurement does not begin until after equilibrium is reached.

enzyme

In assays which measure increasing absorbance, initial absorbance should be below

0.300 abs

In assays which measure decreasing absorbance, initial absorbance should be above

0.900 abs

If false results are suspected because of high enzyme levels, this is the solution

dilution

CK is mostly found in these types of tissue

muscle, brain

The CK enzyme adds groups to _.

phosphate, creatine

These are the specimens of choice for CK analysis

serum or heparin plasma

The CK of hemolyzed blood may be falsely

low

CK levels begin to rise within 1-2 hours of

muscle trauma

Within _ hours of an AMI, CK levels should be above the reference range.

3-4

The two possible subunits of CK are

M, B

CK-1 is

CK-BB

CK-2 is

CK-MB

CK-3 is

CK-MM

The has a higher concentration of CK-MB than other muscles

CK-MM

The CK-MB index ratio is calculated by this equation

CK-MB / CK

A CK-MB above ____% is chemically consistent with an AMI

6

CK-MM, CK-MB, and CK-BB are

oenzymes

The M subunits of CK contain terminal groups which can be

lysine, deaminated

A CK-MM molecule with both subunits deaminated is termed

CK-MM1

A CK-MM molecule with neither subunit deaminated is termed

CK-MM3

CK-MM and MB isoforms can be used to provide an idea of

how long ago muscle damage occured

LDH isoenzymes convert

keto groups, hydroxy groups

LDH's most preferred substrates are

pyruvic acid, lactic acid

The substrate of the LDH Wacker reaction is

lactic acid

The substrate of the LDH Wroblewski reaction is

pyruvic acid

The tissues which contain especially high amounts of LDH are

blood cells, muscles

The LDH of hemolyzed blood may be falsely

high

LDH is the only enzyme which should never be

frozen

LDH has ___ subunits and __ types of subunit

4, 2

LDH 1 & 2 are most commonly found in

blood cells, heart

LDH-3 is most commonly found in

lung

LDH 4 & 5 is most commonly found in

muscle, liver, kidney

Only these LDH isoenzymes will use alpha-hydroxybutyric acid as a substrate

1, 2