Kinetics and mechanism of proteolytic enzyme catalysed reactions : a thesis presented for the degree of Doctor of Philosophy in Biochemistry

KINETICS AND ^{MECHANISM OF}

PROTEOLYTIC ENZYME CATALYSED REACTIONS

A thesis presented for the degree

o! D o c tor of

Phil osophy in Biochemistry

b y

Michael John Boland

June

1 9 73 .

Th e mos t i n c omp re h en s i b le t h ing about the u n i v e r se is i t s c omp reh e n s i b il i t y .

E i n s t e in .

Acknowledgeme n t s

I woul d l ike to t ha nk D r . M.J. Ha rdman f o r h e l p ful a d v i ce ·and d i s c u s s i o n during the· c ours e of this work.

Thanks are als o d u e t o D r. J .W. L y t t l e to n f o r c ar r y i ng

o u t th e ul t ra c e n t r i f uge exp e r ime nt s , D r. I.D. Wa ts o n f o r a dv i c e on t h e p hy s i c o - c h em i cal s ec t ion , D r. M .A . McDowa l l f o r c ommun i ca t io n o f unpubl i s he d r e s ul t s , D r. C.R. Bo s we l l f o r as s i s t anc e i n des ign i n g t he s imula t i on p r o gram ,

Pro f e s s o r ^R. Hodg e s f or c a r r y ing out mas s s pe c t ral

a na l ys e s , and t o th e App l ied B i o c h em i s t ry D i v i s i o n of th e D.S . I . R . f o r a s tu d y awa rd u n d e r wh i ch t h i s work was

c ar r i e d out .

c hem icall y and kinet i call y. 'Th e enzyme ha s man y s t ruc tural and ^kinetic similar i t ies with f ic i n and p a p a i n . Spe ci f i c i t y

s t ud ies indicate a s t r o ng pref e r e n^ce f o r a bas i c s i de c h a i n i n the S 1 s i te, and competitive i n h ibit o r b i nd i ng s h ows a

Ina ct ivati_on p reference f o r an aromatic gr oup i n the S2 s i te.

s tudies s how _{the p}re_se n^c_e of o ne a c t ive thiol g ro up per enzyme ^molecule.

The hyd rolysis o f N�- c a r b o benzoxy-L - l y s i ne E- n i t ro p hen y l este r b y actini d in ha s been s tud ied in det a i l . The Michae l i s

c onstant, K , is dependent on groups i o n i s i ng a t pH 3.75

m

and 8 . 1 . The turnover number, k t ' s h ow s l i t t l e pH

ea

dep e ndence a t low pH but a n upwa rd i n f le ct i o n d ep e n d e n t on a g r oup i o n ising at pH 8.1. When the rea c t i on i s f o l l owed

w i t h enzyme c oncen t ra t ion i n ^excess of s u bs t ra t a c on c ent ra t i on a biphasic reaction is observed. This i s i n t e rpre ted b y a mec ha nism ^similar to that p r op o s ed f o r fi c i n and p apain catalysed hyd rolyses of t_his subs t ra t a . Th is me chnni s m i s m o r e c omp l i cated than the s imple acyla tion-deac yla t ion mecha n i sm no rmall y _expected, i nv o lv i ng an i s o me r i s a tion of s ome kind . Micr oscop i c rate c on s t a n t s f or t he reac t i o n hav e bee n calc ula ted.

The sign ificance of va r i ous p h y sico-chemi c al p r i ncip le s o f c a tal y s i s h a s b e e n d is cus s ed i n rela tio n t o en zymic

c a ta l y s i s . From a s tud y of th e imid a zo l e ca tal y�ed

hyd r o lys i s o f N , O- d ia c e t y l s e r i nam i d e , i t has b e e n

c on c l u d e d t h a t gene ral b a s e c a tal y s is c ould p l a y a much grea t er p a r t i n e n zym i c catalys is than had prev i ou s ly b e e n e s t imated .

Abstract

SECT ION I . GENERAL INTRODUCTION

1. Earl y H i s t o ry o f Enzymology 2 . Proteolytic Enzyme s

SECTION I I . THE PLANT TH IOL PROTEASES

1. I n t roduc t i o n

2. Expe r imental

3. Res u l t s and D i s cus s i on

SECT ION Ill . PHYS ICO-CHEM ICAL PR INC IPLES OF ENZYM IC CATALYSIS

1. I n t r o d uc t i o n 2 . Exp erimental

3. Res u l t s

4. D i s c u s s i on

SECTION IV . CONCLUS ION

1. Prop e r t i e s of Ac t i n i d i n

2. Phy s i c o-Chem i ca l Fa c t ors i n Ac t i n i d i n Ca tal ys od Hydro l ys is o f Z- l y s - pNP

Appendix 1.

Ap pen^dix 2.

Bibliography

Computer Programs

D e r ivat i o n of K i ne t i c Parameters f o r

Act1n1d1n Catalysed H^ydrolysis of

Z- l y s-pNP

Page

V

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15 30 46

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Li st of Figures

1. Ac t iv i t y + P ro t e i n Concentrat io n Prof i l e s f o r

E l ut io n o f Act i n idin from H i gh R e s o l u t i o n I on Exchange Ch romatograph y.

2 . P l o t s f or D e t e r m i nat ion of the Mol ecul a r We i ght of Ac t in i d i n .

3 .

Ef f ec t of Compe t i t ive Inh ibi t o rs on Ac t i n id i n Cata l y s ed H y d r o l ysis _of Z-l�s -pNP.

4. L i neweav e r-Burk p l ots for A c t i n i d i n Ca ta l y s e d Hyd ro l y s i s of A l k y l Est ers.

5 . pH Prof i l e f or k t•

e a

6. pH Profi l e f o r K •

m

7. O s c i l l o s c o p e Tra ce f or Act i n id i n Catal y s ed

48

50

56

5 8 6 6 67

Hydro l y s i s of Z-l ys-pNP w i th Enzyme in Exc es s . ⁷⁰

s. Dep enden c e of Kb on Enzyme Concen t rat i o n . 73

9 .

Doub l e R e c i p r o c a l Plot of Dep end ence of Bur s t

··S i z e o n E n zyme Concentration .

7 7

10. Compa r i s o n o f Act ua l and Simu l ated Resu l t s f or

Ac t i n i d i n Cata l ys ed Hydro l ys i s of Z- l ys -pNP. ^. 84

1 1 . D e p end e nc e o f Rate of H y d ro l ys is o f N , O , D iac e t y l S e r i nam1d e o n Im i d az o l e Conc ent ra­

t i on .

1 2. A rrhen i u s Pl o t fo r Imid a z o l e Catalys e d H y d rol ys i s o f N ,O , D ia c e t y l Se r i nam id e .

1 3 . S c h emat i c D i agram o f S imu l a t ion Prog ram .

1 0 0

1 0 2 1 1 7