FREE ENERGY

BY THE SECOND LAW NO PHYSI CAL OR CHEMI CAL PROCESS CAN BE 100%
EFFI CI ENT. THUS USABLE ENERGY MUST DECREASE.
TOTAL ENERGY = USABLE ENERGY + UNUSABLE ENERGY
TOTAL ENERGY ENTHALPY OR ~H
USABLE ENERGY FREE ENERGY OR ~G
UNUSABLE ENERGY DISORDER OR ENTROPY (~S) TIMES TEMPERATURE (~TS)
SI NCE WE ARE I NTERESTED I N THE CHANGE I N AVAI LABLE USABLE ENERGY
DURI NG A PROCESS I T I S COMMON TO REARRANGE THE RELATI ONSHI P AND
EXPRESS I T AS
G = H - TS
FOR A CHEMI CAL REACTI ON THI S REPRESENTS THE DI FFERENCE I N FREE
ENERGY OF THE PRODUCTS AND REACTANTS.
SYSTEMS CAN SPONTANEOUSLY MOVE FROM A HI GHER TO A LOWER FREE
ENERGY STATE. LESS USABLE ENERGY RESULTS I N A MORE ~STABLE STATE
i.e. A STATE LESS LI KELY TO UNDERGO FURTHER CHANGE. A CHEMI CAL
REACTI ON AT I TS LOWEST ENERGY STATE I S SAI D TO BE I N EQUI LI BRI UM.
G NEGATI VE - FREE ENERGY I S RELEASED
G POSI TI VE - FREE ENERGY I S REQUI RED TO PROMOTE THE REACTI ON
G I S ZERO - THE REACTI ON I S AT EQUI LI BRI UM
ACTI VATI ON OF CHEMI CAL REACTI ONS
SPONTANEOUS CHEMI CAL REACTI ONS MAY NOT OCCUR AT A RAPI D RATE.
THI S I S A RESULT OF THE FACT THAT REACTI ONS REQUI RE THE BREAKI NG AND/OR
FORMATI ON OF (moderately stable) BONDS BETWEEN THE ATOMS.
THI S GENERALLY I NVOLVES CONTORTI NG ONE OR MORE BONDSI N THE STARTI NG
MOLECULE I NTO AN UNSTABLE (energetic) STATE BEFORE THE REACTI ON CAN
I NI TI ATE.
TO REACH THI S CONTORTED STATE REACTANT MOLECULES MUST ABSORB ENERGY
FROM THEI R SURROUNDI NGS.
THE CONTORTED STATE I S SOMETI MES REFERRED TO AS THE ACTI VATED
~TRANSI TI ON STATE.
TO ENHANCE THE NUMBER OF MOLECULES IN THE ~TRANSITION STATE AND THUS
ACCELERATE THE REACTI ON CHEMI STS OFTEN SUPPLY THERMAL ENERGY
(heat the reaction) TO I NCREASE THE SPEED OF MOLECULAR MOTI ON AND THUS THE
AMOUNT OF ENERGY I NVOLVED I N MOLECULAR COLLI SI ONS.
ALTERNATI VELY THEY MAY SUPPLY A CATALYST. THI S I S A CHEMI CAL OR PHYSI CAL
ENTITY WHICH CAN ~INTERACT WITH THE REACTANT IN SUCH A FASHION THAT THE
BONDS I N THE REACTANT ARE DI STORTED.
ENZYME CATALYSTS
SPONTANEOUS REACTI ONS MAY STI LL PROCEED SLOWLY DUE TO AN ENERGY
BARRIER, THE ~ACTIVATION ENERGY, WHICH MUST BE EXCEEDED.
ENZYME CATALYSTS CAN LOWER THI S BARRI ER AND ACCELERATE THE REACTI ON
CATALYSTS DO NOT AFFECT THE OVERALL ENERGY CHANGE I N A REACTI ON
AND THUS CANNOT FORCE AN UNFAVORABLE (ENERGY REQUI RI NG) REACTI ON
TO OCCUR. WHEN ALL AVAI LABLE CATALYST I S I NVOLVED, THE MAXI MUM RATE
OF REACTI ON I S SEEN.
The Discovery of Enzymes
IN THE MID 1800`S A DEBATE AROSE AS TO THE ORIGEN OF ORGANIC
MOLECULES.
~VITALISTS (e.g. Pasture) CLAIMED LIVE CELLS WERE REQUIRED.
~MECHANISTS SUGGESTED THAT COMPONENTS WITHIN CELLS
CARRI ED OUT THE SYNTHESI S AND THAT LI FE PER SE WAS NOT
I MPORTANT.
I N 1897 THE DEBATE WAS RESOLVED I N FAVOR OF THE MECHANI STS
WHEN EDUARD BUCHNER SHOWED THAT SUGAR COULD BE CONVERTED
I NTO ALCOHOL BY A CELL-FREE EXTRACT FROM YEAST.
THE COMPONENT INVOLVED WAS THEN NAMED AN ~ENZYME (which
means ~in yeast) BY F.W. KUHNE. (Note: the ~enzyme you will study in lab
will be derived from classical yeast extracts)
THE FI ELD OF ENZYME STUDI ES WAS ADVANCED CONSI DERABLY
BETWEEN 1926 AND THE EARLY 30`S WHEN 1AMES SUMNER SUCCEDED
I N I SOLATI NG, PURI FYI NG AND CRYSTALI ZI NGTHE FI RST SUCH
BI OLOGI CAL CATALYST, UREASE, AND SHOW THAT I T WAS A PROTEI N.
IT WAS ALSO DURING THIS EARLY PEIOD OF THE 30`S THAT 1.B.S. HALDANE
MADE A THE REMARKABLE SUGGESTI ON THAT WEAK-BONDI NG
I NTERACTI ONSBETWEEN AN EZZYME AND I TS SUBSTRATE MI GHT BE
USED TO DI STORT THE SUBSTRATE AND CATALYZE A REACTI ON.
THI S I NSI GHT GUI DED MUCH RESEARCH AND I S THE FOUNDATI ON
OF OUR PRESENT UNDERSTANDI NG OF ENZYME ACTI ON.
THE MODERN CLASSI FI CATI ON OF ENZYMES
AS THE STUDY OF BIOCHEMISTRY PROCEEDED THROUGH THE 1900`S AN EXTENSIVE ARRAY OF ENZYMES
WERE DI SCOVERED. TO BRI NG SOME ORDER TO THE FI ELD THEY HAVE BEEN DI VI DED I NTO 6 CLASSES,
EACH OFEN WI TH SUB-GROUPI NGS.
NOTES:
1. MOST OXIDOREDUCTASES ARE REFERRED TO AS ~DEHYDROGENASES
2. A HYDROLASE I S SOMETI MES CONSI DERED A SPECI AL CLASS OF TRANSFERASE
3. A LYASE THAT CATALYZES AN ADDITION REACTION IN CELLS IS OFTEN CALLED A ~SYNTHASE
4. LIGASES, ALL OF WHICH REQUIRE AN ENERGY INPUT, ARE USUALLY REFERRED TO AS ~SYNTHETASES.
ENZYMES
ENZYMES ARE USUALLY PROTEINS. THEY BIND THEIR ~SUBSTRATES
(REACTANTS) AT A SPECIALLY DESIGNED ~ACTIVE SITE. THIS DISTORTS THE
SUBSTRATES INTO A HIGHER ENERGY ~TRANSITION STATE, WHICH PROMOTES
THE TRANSITION OVER THE ~ACTIVATION ENERGY BARRIER.
ENZYME/SUBSTRATE I NTERACTI ON AT THE BI NDI NG POCKET
THE BI NDI NG REGI ON OF THE ACTI VE SI TE OFTEN USES MULTI PLE
NON-COVALENT INTERACTIONS TO ENGAGE THE ~LIGAND.
TWO VARIETIES ARE CONSIDERED; ~LOCK AND KEY, OR ~INDUCED FIT.
NOTE: these terms might be misleading - L&K is usually used for an average enzyme to indicate specificity and I nduced Fit
to describe the action of a regulated enzyme.
IN ACTUALITY ALL ENZYMES ACT THROUGH A VARIATION ON ~INDUCED FIT. THE SHAPE OF THE
BI NDI NG POCKET I S NOT A PRECI SE MATCH TO THE SUBSTRATE. RATHER, I T I S SUFFI CI ENTLY CLOSE
TO I NI TI ATE BI NDI NG AT A LI MI TED NUMBER OF SI TES. THI S BI NDI NG WI LL DI STORT BOTH THE SUBSTRATE
AND THE ENZYME (Koshland), ACTI VATI NG FURTHER BI NDI NG SI TES, AND THUS FURTHER DI STORTI ON.
THE BEST L&K BINDING IS TO THE ~INDUCED SUBSTRATE TRANSI TI ON STATE CONFI GURATI ON (L. PAULI NG
1946). ONCE I N THAT STATE THE SUBSTRATE CAN READI LY CONVERT I NTO PRODUCT(or return to the starting state).
I T I S THE SEQUENTI AL RELEASE OF BI NDI NG ENERGY THAT CONTRI BUTES TO THE REDUCTI ON OF THE
ACTI VATI ON ENERGY OF THE REACTI ON AND PROMOTES CATALYSI S.
THE ACTIVE SITE
THE CATALYTI C REGI ON OF THE ACTI VE SI TE MAY USE TRANSI ENT
NONCOVALENT OR COVALENT I NTERACTI ONS WI TH THE SUBSTRATE TO
DISTORT BONDS TO THE ~TRANSITION STATE THEREBY LOWERING
THE ACTI VATI ON ENERGY BARRI ER.
ENZYME KI NETI CS
CONSI DER THE SI MPLE REACTI ON OF THE CONVERSI ON OF ONE CHEMI CAL
TO ANOTHER. S P
THI S COULD BE ACCELERATED BY THE USE OF AN ENZYME:
S + E ES E + P
SI NCE THE ANALYSI S OF TWO BODY REACTI ONS CAN BE COMPLEX, ESPECI ALLY
WHEN THE CONCENTRATI ONS OF THE COMPONENTS ARE CHANGI NG, A
SI MPLI FYI NG APPROACH I S USED. KENETI C EXPERI MENTS USUALLY MEASURE
~I NI TI AL VELOCI TY (V
0
) WHEN [S] I S MUCH GREATER THAN [E] AND CAN
ESSENTI ALLY BE CONSI DERED A CONSTANT. THUS THE REACTI ON ABOVE CAN
BE CONSI DERED A (PSEUDO)-FI RST ORDER REACTI ON, WI TH THE RATE
DEPENDENT ONLY ON THE STARTI NG [S]. AS SUCH AS [S] RI SES THE RATE WI LL
I NCREASE UNTI L THE ENZYME I S SATURATED, AT WHI CH POI NT A MAXI MUM
VELOCI TY Vmax WI LL BE REACHED.
THI S ALSO I MPLI CTES THE ES COMPLEX AS A CRI TI CAL I NTERMEDI ATE I N THE
ANALYSI S OF THE ENZYMATI C REACTI ON. I N 1913 MI CHAELI S AND MENTEN
EXPANDED ON THI S I DEA TO PRODUCE A GENERAL THEORY OF CATALYSI S,
WHI CH WAS AMENABLE TO MATHMATHI CAL ANALYSI S. THEY POSTULATED:
THE FI RST STEP WAS THE RAPI D AND REVERSI BLE FORMATI ON OF AN
ENZYME/SUBSTRATE COMPLEX.
k
1
E + S ES
k-
1
THEN ES BREAKS DOWN AT A SLOWER SPEED (i.e. rate limiting) TO FORM PRODUCT.
k
2
ES E + P
k-
2
ENZYME KI NETI CS
THE ENTI RE VERSI ON OF THE MI CHAELI S/MENTEN EXZYME REACTI ON ANALYZED FOR I NI TI AL RATES I S;
k
1
k
2
E + S ES E + P
k-
1
k-
2
WHEN EXAMI NI NG I NI TI AL RATES THE k-
2
TERM CAN BE I GNORED (P is negligible at the reaction start) AND
SI NCE ES BREAKDOWN I S LI MI TI NG -
V
0
= k2[ES]
WHAT WE WI SH TO BE ABLE TO ANALYSE I S THE I NI TI AL VELOCI TY (V
0
), WHI CH I S NOW SEEN TO BE
DEPENDENT UPON THE CONCENTRATI ON OF THE ENZYME/SUBSTRATE COMPLEX - A PROPERTY THAT I S
NOT EASI LY MEASURED. THUS WE I NTRODUCE Et - THE TOTAL ENZYME CONCENTRATI ON, WHI CH ALLOWS
US TO DEFI NE THE CONCENTRATI ON OF FREE ENZYME [E] AS = [Et] - [ES]. NOWWE CAN ATTEMPT TO
SOLVE THE EQUATI ON FOR V0 USI NG EASI LY MEASURED PARAMETERS.
STEP 1. DEFI NE THE RATES OF FORMATI ON AND BREAKDOWN OF ES
RATE OF ES FORMATI ON = k
1
([Et] - [ES])[S]
RATE OF ES REAKDOWN = k-
1
[ES] = k
2
[ES]
STEP 2. AN I MPORTANT ASSUMPTI ON, I NTRODUCED BY LI NEWEAVER AND BURK, I S THAT THE I NI TI AL
RATE OF REACTION REFLECTS A ~STEADY STATE, IN WHICH ES] IS CONSTANT i.e. its rate of
formation and breakdown are equal.
THI S THEN RESULTS I N THE EXPRESSI ON:
k
1
([Et] - [ES])[S] = k-
1
[ES] + k
2
[ES]
ENZYME KI NETI CS
k
1
([Et] - [ES])[S] = k-
1
[ES] + k
2
[ES]
STEP 3. SOLVE THE ABOVE EQUATI ON FOR [ES] VI A ALGEBRAI C MANI PULATI ON.
MULTI PLY OUT LEFT AND SI MPLI FY RI GHT
k
1
[Et][S] - k
1
[ES][S] = (k-
1
+ k
2
)[ES]
ADD k1[ES][S] TO BOTH SI DES AND SI MPLI FY
k
1
[Et][S] = (k
1
[S] + k-
1
+ k
2
)[ES]
NOW SOLVE FOR [ES]
[ES] = k
1
[Et][S]
k
1
[S] + k-
1
+ k
2
SI MPLI FY BY COMBI NI NG RATE CONSTANTS ( i.e. multiply/divide both sides by unity as 1 or k
1
/k
1
[ES] = [Et][S]
[S] + (k
2
+ k-
1
)/k
1
THE COMBINED RATE CONSTANTS ARE NOW DEFINED AS THE ~MICHAELIS CONSTANT OR Km.
SUBSTI TUTI NG THAT GI VES:
[ES] = [Et][S]
Km + [S]
ENZYME KI NETI CS
[ES] = [Et][S]
Km + [S]
STEP 4. WE CAN NOW SOLVE THE ~INITIAL RATE EQUATION - V
0
= k
2
[ES] USI NG OUR NEW [ES] VALUE
V
0
= k
2
[Et][S]
Km + [S]
REMEMBER THAT THE MAXI MUM VELOCI TY OF AN ENZYME REACTI ON WI LL OCCUR WHEN THE
ENZYME I S SATURATED WI TH SUBSTRATE, i.e. WHEN [ES] = [Et]. EXPRESSED KI NETI CALLY Vmax = k2[Et]
SUBSTI TUTI NG THI S I N THE ABOVE EQUATI ON WE HAVE
V
0
= Vmax[S]
Km + [S]
THI S I S THE MECHAELI S-MENTEN EQUATI ON, EXPRESSED I N TERMS THAT CAN BE EXPERI MENTALY
DETERMI NED AND/OR MATHMATI CLLY ANALYSED.
ENZYME KI NETI CS - GRAPHI CAL REPRESENTATI ON
THE MEANI NG OF Km:
1. HERE I T CAN BE SEEN THAT I T I S
THE SUBSTRATE CONCENTRATI ON
AT THE MAXI MAL VELOCI TY.
2. MATHMATI CLLY I T I S THE RATI O
OF THE RATE CONSTANTS FOR
BREAKDOWN DI VI DED BY THOSE
FOR FORMATI ON OF THE ES
COMPLEX.
Km = k
2
+ k-
1
k
1
DEPENDI NG ON THE ENZYME, AND
SUBSTRATE I NVOLVED THI S CAN BE
A COMPLEX AND CHANGEBLE ENTI TY
3. I N CASES WHERE k
2
I S RATE LI MI TI NG
k
2
<< k-
1
Km REDUCES TO k
2
/k
1
.
THI S WOULD BE THE DI SSOCI ATI ON
CONSTANT FOR THE ENZYME, AND
I N THESE LI MI TED CASES A MEASURE
OF THE ENZYME`S AFFI NI TY FOR THE
SUBSTRATE.
ENZYME KI NETI CS - THE LI NEWEAVER-BURK EQUATI ON
ENZYME I NHI BI TI ON
ENZYME I NHI BI TORS ARE MOLECULAR AGENTS THAT I NTERFERE WI TH CATALYSI S, SLOWI NG OR
HALTI NG ENZYMATI C REACTI ONS. AS SUCH THEY ARE OFTEN USED TO STUDY THE MECHANI SM
OF ENZYME ACTI ON. ALSO SI NCE ENZYMES CATALYZE VI RTUALLY ALL CELLULAR PROCESSES,
I T I S NOT SURPRI SI NG THAT SUCH I NHI BI TORS ARE AMONG THE MOST I MPORTANT
PHARMACEUTI CAL AGENTS KNOWN.
THE MAI N VARI ETI ES OF ENZYME I NHI BI TI ON I NCLUDE;
COMPETI TI VE
UNCOMPETI TI VE AND
MI XED OR NONCOMPETI TI VE
THESE VARI ETI ES OPERATE BY DI FFERENT MECHANI SMS AND HAVE DI FFERENT AFFECTS ON THE
KI NETI C PARAMETERS OF THE ENZYMES I NVOLVED.
COMPETI TI VE I NHI BI TI ON
CATALYTI CALY COMPETENT - GREEN
I NCOMPETENT - RED
COMPETI TI VE I NHI BI TORS ARE THE MOST COMMON.
USUALLY THEY RESEMBLE THE SUBSTRATE AND THUS COMPETE
WI TH I T FOR BI NDI NG TO THE ACTI VE SI TE i.e. they only bind to enzymes
with an empty active site.
THE FORMATI ON OF EI CAN BE REVERSED BY SUFFI CI ENTLY HI GH
CONCENTRATI ONS OF SUBSTRATE. THUS Vmax I S UNCHANGED.
HOWEVER, THE SUBSTRATE CONCENTRATI ON AT Vmax I S
I NCREASED i.e. Km I S RAI SED. (The measured Km, here is called the
apparent Km (Km), where is the proportion by which the Km is altered.)
UNCOMPETI TI VE I NHI BI TI ON
COMPETENT - GREEN
I NCOMPETENT - RED
AN UNCOMPETI TI VE I NHI BI TOR BI NDS AT A SI TE DI STI NCT FROM
THE ACTI VE SI TE, AND ONLY TO THE ES COMPLEX, NOT TO FREE
ENZYME. SI NCE I T I S THE ES COMPLEX THAT BI NDS I Vmax I S
DECREASED BY THE CONVERSI ON OF SOME MOLECULES OF E TO
THE I NACTI VE ESI FORM, A RESPONSE THAT CANNOT BE REVERSED
BY ADDI TI ON OF MORE S. UNCOMPETI TI VE I NHI BI TORS ALSO
DECREASE Km SI NCE I SHI FTS THE EQULI BRI UM TOWARD ENZYME
COMPLEX FORMATI ON. (NOTE: the Vmax obtained here is referred to as
the apparent Vmax or `Vmax.)
MI XED I NHI BI TI ON
COMPETENT - GREEN
I NCOMPETENT -RED
A ~MIXED INHIBITOR , LIKE THE UNCOMPETITIVE, ALSO BINDS AT
A SI TE DI STI NCT FROM THE ACTI VE SI TE. HOWEVER, I T DOES SO
WI TH EI THER FREE ENZYME (E) OR WI TH THE ENZYME/SUBSTRATE
COMPLEX (ES), FORMI NG I NACTI CE EI OR ESI COMPLEXES.
USUALLY, MI XED I NHI BI TORS AFFECT BOTH Km AND Vmax.
A special case (rarely seen) is NONCOMPETI TI VE I NHI BI TI ON. Here, the
relative affect on Km (like competitive inhibitors) is equal to the relative
effect on Vmax(like uncompetitive inhibitors), (=`) with the result that only the
apparent Vmax is altered, not the apparent Km, as seen in the Lineweaver-Burk
graph below.
Regulation of Enzyme Activity
Allosteric regulation is the process wherein enzyme
activity is controlled by the binding of small
molecules to regulatory sites on the enzyme.
Phosphorylation is a particularly common
mechanism for regulating enzyme activity.
The addition of phosphate groups either stimulates or
inhibits the activities of many different enzymes.
ALLOSTERI C REGULATI ON
HERE A CONFORMATI ONAL CHANGE I S I NDUCED I N THE PROTEI N BY THE
BI NDI NG OF A NON-SUBSTRATE CHEMI CAL TO THE ENZYME AT A POSI TI ON
REMOTE FROM THE ~ACTIVE SITE. THE RESULTING CHANGE CAN EITHER
ACTI VATE OR I NHI BI T THE ENZYME. FEEDBACK I NHI BI TI ON OFTEN EMPLOYS
THI S REGULATORY METHOD.
FEEDBACK REGULATI ON
ENZYME REGULATI ON VI A PHOSPHORYLATI ON
SINCE THIS PROCESS IS ITSELF CONTROLLED BY AN ENZYME, MANY ~PRODUCT
ENZYMES CAN BE PRODUCED. THI S LEADS TO LARGE SCALE CHANGES I N ACTI VI TY.
THE RESPONSIBLE ~KINASES ARE OFTEN CONTROLLED BY SIGNALING PROCESSES.