The following experimenal daa daa were colleced during a sudy of he caalytc actiy of an inestnal peptdase capa!le of hydroly"ing he dipeptde glycylglycine# Glycylglycine $ H%& ' % glycine
()* +m,1#5 %#2 3#2
.roduc formed +/mol0min2 #% 1 2 #% 2 #% 4
()* +m,#2 4#2 1#2
.roduc formed +/mol0min2#33 2#2 2#5
6rom hese daa deermine !y graphical analysis +see 7ox 481- he alues of K m and V max max for his en"yme preparaton and su!srae# 2.
,any en"ymes are inhi!ied irreersi!ly !y heay8meal ions such as Hg%$ Cu%$ or :g$ which can reac wih essental sul;ydryl groups o form mercaptdes< =n">)H $ :g$ ' =n">)>:g $ H $ The a?niy of :g$ for sul;ydryl groups is so grea ha :g $ can !e used o trae @)H groups Auantately Auantately## To 12 mB of a soluton conaining 1#2 mg0mB of a pure en"yme was added us enough :gDo3 o compleely inactae inactae he en"yme# : oal of 2#3% /mol of :gD& :gD&3 was reAuired# Calculae he minimum molecular weigh of he en"yme# Ehy does he alue alue o!ained in his way gie only he minimum molecular weighF
3.
The en"ymatc actiy of lyso"yme is optmal a pH 5#% +see graph !elow-# The acte sie of lyso"yme conains conains wo amino acid residues essental for caalysis< caalysis< Glu35 and :sp5%# The pK a alues of he car!oxyl side chains of hese wo residues are 5#9 and #5 respectely# respectely# Eha is he ioni"aton sae +proonaed +proonaed or deproonaed- of each residue a he pH optmum of lyso"ymeF How can he ioni"aton saes saes of hese wo amino acid residues explain he pH8 actiy prole of lyso"yme lyso"yme shown !elowF
1
4.
Two dieren en"ymes are a!le o caaly"e he same reacton : ' 7# They !oh hae he same V max !u dier in heir K m for he su!srae :# 6or en"yme 1 he K m is 1#2 m,I for en"yme % he K m is 12 m,# Ehen en"yme 1 was incu!aed wih 2#1 m, : i was o!sered ha 7 was produced a a rae of 2#22%2 mmoles0minue# a. b. c.
5.
Eha is he alue of he V max of he en"ymesF Eha will !e he rae of producton of 7 when en"yme % is incu!aed wih 2#1 m, :F Eha will !e he rae of producton of 7 when en"yme 1 is incu!aed wih 1, +i#e# 1222 m,- :F
:n en"yme can caaly"e a reacton wih eiher of wo su!sraes )1 or )%# The K m for )1 was found o !e %#2 m, and he K m for )% was found o !e %2 m,# : suden deermined ha he V max was he same for he wo su!sraes# Jnforunaely he los he page of his noe!ook and needed o know he alue of V max# He carried ou wo reactons< one wih 2#1 m, )1 he oher wih 2#1 m, )%# Jnforunaely he forgo o la!el which reacton u!e conained which su!srae# eermine he alue of V max from he resuls he o!ained< Tu!e Dum!er 1 %
6.
Lae of formaton of produc 2#5 #4
:n en"yme caaly"es a reacton a a elociy of %2 /mol0min when he concenraton of su!srae +)- is 2#21 ,# The K m for his su!srae is 1 x 1285 ,# :ssuming ha ,ichaelis8,enon kinetcs are followed wha will he reacton elociy !e when he concenraton of ) is +a- 1 x 1285 , and +!- 1 x 12 8 ,F
%
7.
:n en"yme caaly"es he reacton : ' 7# The inital rae of he reacton was measured as a functon of he concenraton of :# The following daa were o!ained< (:* micromolar 2#25 2#1 2#5 1 5 12 52 122 522 1222 5222 12222 %2222
V 2 nmoles0min
2#24 2#1 2#M9 1# M#3 13 2 53 M3 M M9 42 42
7e sure o include unis in your answers# Eha is he K m of he en"yme for he su!srae :F b. Eha is he alue of V 2 when (:* N 3F The a!oe daa was ploOed as 10 V 2 s# 10(:* and a sraigh line was o!ained# c. Eha is he alue of he y8inercep of he lineF d. Eha is he alue of he x8inercep of he lineF a.
8.
Ehen 12 /g of an en"yme of Mr 52222 is added o a soluton conaining is su!srae a a concenraton one hundred tmes he K m i caaly"es he conersion of M5 /mol of su!srae ino produc in 3 min# Eha is he en"ymePs urnoer num!erF
9.
Q has !een proposed ha for en"ymes ha o!ey ,ichaelis8,enon kinetcs he K m alue for an en"yme may represen he approximae physiological su!srae concenraton in he cell# =xamine a ypical plo of V ersus ()* and explain why his appears o !e a reasona!le suggeston#
