Conformation of pyridine nucleotide coenzymes by rotary dispersion and dichrois

Stryer, L.

English

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. c . 
PROGRESS REPORT 
TO THE 
NATIONAL AERONAUTICS AND SPACE ADiMINISTKATION 
R e s e a r c h  Grant  NGR-05-020-  137: S t ruc tu re  and Funct ion of Protei l ls  alld 
PI- l_-.-_L- 
Nucleic Acid -- 
Semi-annual  Repor t  f o r  the Pe r iod  July 1, 1967 to December 31, 1967 ---- 
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ff 653 July65 
Depar tment  of Biochemistry 
School of Medicine 
Stanford Uiiive r s ity 
P a l o  Alto, Cal i fornia  
I 
Luber t  S t r y e r ,  M. D. 
P r inc ipa l  Investigator 
https://ntrs.nasa.gov/search.jsp?R=19680011670 2020-03-12T08:34:17+00:00Z
NGK - 05 - 02 0-  1 37 
Pagc  One 
I. P U  B LI C A TI ONS 
A. During the Y e a r  of 1967 - -I 
1. Haugland, R. P. , Stryer ,  L. ,  Stenglc, T. K .  and Baldeschwieler,  
J. D. , NMR Studies of Antibody-Hapten Interactions Using a Chloride Ion 
P robe ,  Biochemistry,  6 ,  498, (1 567)  - 
2. S t rye r ,  1,. , Flolmgrcn, A. and Reichard,  P., Thioredoxjn.  
A Localized. Conformational Change Accompanying Rcduction of the P ro te in  
to thc Sulfhydryl For in ,  Biochemistry,  6,  101 6, (1 967) - 
1- 
3. - Hunclley, L. , Coburn, T . ,  Garwin, E. ' and St ryer ,  L, , A Naiio- 
second F luor ime te r ,  Rev. Sci. Instru, , 38, 488, (1967) -
> 
4. Haugland, R. P. and Stryer ,  L., A Fluorescen t  P r o b e  a t  the 
Active Site of a-Cliymoti-ypsin, i n  "Conformation of Biopolymers,  ed. 
by G. N. l iamachandran,  Academic P r e s s ,  Vol. 1,  pp. 321-335, (1967) 
5. S t ryer ,  L. and I-laugland, R. P., Energy  Transfer :  A Spectro-  
scopic  Ruler ,  Proc .  Nat'l. Acad. Sci. , 58, 719, (1967) 
_I 
B. During the Yea r  of 1968 
1. Epstein,  1-1. F. and Stryer ,  L. , Kinetics of Azide Binding to 
Normal  and Mutant F e r r i  Henloglobins as Evidence for  Subunit Interaction, 
J. Mol. Biol. ,  31, (1968) 
I 
2, S t rye r ,  L. , Implications of X-Ray Crystal lographic  Studies of 
P ro te in  Structure ,  Ann. Rev. Biochem. , 38, (1968), in  p r e s s  -
3. Galley, W. C. and Strycr ,  L. , Trip le t  - Trip le t  Energy  T r a n s f e r  
in  P ro te ins  as a Cr i te r ion  of Proximity,  Proc. Nat'l.. Acad. Sci . ,  (in p r e s s ,  
1968) 
11. TRIPLET - SINGLET ENERGY TRANSFER IN PROTEINS 
W e  have observed a new typc of encrgy t r ans fe r  in  proteins. 
Tr ip le t  - single't energy t r ans fe r  was exhibitcd by a complex of (;;-chymo- 
t rypsin and proflavin, a chromophoric inhibitor. 
energy  of jndolc groups in the e n z y m e  was t r a n s f e r r e d  to the chromophoric  
inhjbjtors a t  the active site. 
The  t r a n s f c ~ .  was cspressccl  in  t e r m s  of a delayed f luoresccnce of the pro-  
flavine in the nii1lcsecond t ime  range. 
be A useful adjunct t o  s i n g l ~ t  - singlet t r ans fe r  i n  measur ing  dis tanccs  in  
the 15 - 6 0  8 rangc. 
The  t r iplet  escj ta t ion 
T h e  cfficiency of t r ans fe r  was about 757'0. 
Triplet  - sinslct  t r ans fe r  should 
-. * .. . . 
NGR-05-020-137 
Page  TWO 
The conformation of thc coenzymes P-DPN,  P-DPNH, the i r  
i s o m e r s ,  analogues and re la ted  model conipouncls has  been studied by optjcal 
rotatory d ispers ion  (ORD) and c i rcu lar  d ichro ism (CD) measurements .  
1957, Weber f i rs t  suggested the existence of a s tacked s t ruc tu re  fo r  P-DPNH, 
to  account fo r  the f luorescence energy t r a n s f e r  p rope r t i e s  , and the changes 
in  a b  s o r p 1 i on s p c c t r um on p y r o pho s ph a t a s e h y d r o 1 y s i s . 
the l a t t e r  shows that there  is l i t t le hypochroimicity of the 260  m p  bancl re la t ive 
to the 340 m p  band in  the- intact  s t ruc ture ,  but that both bands a r e  hypo- 
chromic  relat ive to A M P  and p-NMNI-I respectively.  
adenine and nicotinamide bases  in  P - D P N  and P-DPNH i s  most  c l ea r ly  shown 
by ORD, and CD. 
not the sums of the separa te  par ts .  
t a s e  hydrolysis ,  and in  propylene glycol. The r e l e a s e  of hypochromjcity 
by these t rea tments  suggests  that c lose- range ,  stacking in te rac t ions  a r e  
indeed involved, 
In 
R e i nve s t i  gat i o n of 
The interact ion of the 
The proper t jes  of the coenzymes in  aqueous solutjon a r e  
Marked changes occur  on pyrophospha- 
S imi la r  r e su l t s  have been obtained with the osidised and reduced 
f o r m s  of the Cz(-isomers , and of the coenzyme analogues containing desamino-  
adenine, 3-acetyl pyridine, 3-pyridine aldehyde and thionicotinamicle. These  
compounds do not in genera l  show singlet  - singlet  energy t r ans fe r  f r o m  
adenine to the reduced nicotinamide moiety, implying that f o r  P - D P N H  the 
geometr ica l  proximity of the two halves is not solely responsible  for  the 
effect. 
F r o m  purely s terochcmical  considerat ions , the molecules  could 
ex i s t  in  many ro t amer i c  conformatioiis. 
s e e n  in  the CD spec t r a  of the osidised coenzymes suggests  the exis tence of 
specif ic  conformations,  and relative or ientat ions of the bases .  This  effect 
is even more  pronounced i n  the coenzyme analogues,  5',  5' diadenosine 
pyrophosphate, and 5' ,  5' diadenosine triphosp'iates, Both show a c l a s s i ca l  
s t rong  exciton spli t  in the CD in the region of the adenine absorption, 
cha rac t e r i s t i c  of a specific a symmet r i c  conformation. 
The  cha rac t e r i s t i c  exciton spli t  
IV. PROPERTIES OF MUTANT €IEMOGLOBINS 
The  reaction of azide ion with ferr ihemoglobin is  30-fold s lower 
than with ferrimyglobin.  
fe r r ihemoglobins  was investigated to dctermine whether the lower reac t ion  
r a t e  of Hb A is due to subunit iiiteractions. 
studied were Hb MI , in  which the p ros ima l  heme-linked histidine res idue  
of each  a-chain is replaced by tyrosine,  and FIS M H ~ ,  in  which the s a m e  
change occur s  i n  the P-chains. 
no rma l  subunits bind azidc ion. 
with azide ion, while I Ib  M k l p  reacts  at  the s lower r a t e  cha rac t e r i s t i c  of 
I-Ib A. 
azide ion. 
The binding of azide ion to two mutant human 
The  abnormal  hemoglobins 
In these mutant hemoglobins,  only the two 
Hb M1 , l ike myoglobin, r eac t s  rapidly 
In cont ras t ,  the fou r  proteins have s imi l a r  binding affinities fo r  
The la rge  difference in reaction r a t e  between I-Ib M I  and IIb A 
a. 
-. . 
. . .  NGR - 0 5 - 02 0- 1 37 
Page  T h r e e  
IV. P rope r t i e s  of hlIutan'i €Ieimoglobins - continued 
shows that the binding kinetics of the P-chain depend on whethcr the adjacent 
subunit i s  normal  or mutant. Differences i n  absorpt ion s p e c t r a  in  the Sore t  
. region of the azide der ivat ives  of I I b  h41 and Hb M H ~ ,  as well a s  differences 
in  their  r a t e s  of react ion with azide ion, indicatc that c losely r e l a t ed  
s t ruc tu ra l  changcs in the u and /3 chains can have d i s s imi l a r  effects.  
S U B U N I T  I N T E K A C  TION J.N TRY PTOPI-JAN SYNTHET'ASE ---- I__- v. - 
Tryptophan synthetase is an  enzyme composed of two types of 
The  /32 protein contains two pyridoxal 
readi ly  separable  protein subunits, CX and P2. The fully assoc ia ted  
enzyme has  the s t ruc tu re  ctz p2. 
phosphate res idues  that s e r v e  as prosthet ic  groups in  the reac t ions  involving 
L - s e r i n e ,  one of the subs t ra tes .  
coenzyme make i t  a very  useful probe of the act ive 'site of this  enzyme. 
have observed  a highly specif ic  f luorescence emiss ion  f r o m  the pyridoxal 
phosphate groups i n  a complex of the p2 protein and L-ser ine .  Related 
compounds such as  D-ser ine ,  o -ace ty l -L-ser ine ,  DL-ho~nose r ine ,  L- 
Threonine,  and L-alanine a r e  ineffective in  elicit ing the emiss ion ,  showing 
that the interact ion has  a high degree of sterospecificily.  Binding of indole, 
the second subs t ra te ,  diminishes  the f luorescence intensity. Most interest ing,  
binding of t heekubun i t  diminishes the f luorescence intensity;  this  spec t ro -  
scopic  change is co r re l a t ed  with a change in  enzymatic activity. 
l ikely that the emitt ing spcc ies  i s  an intermediate  in the reac t ion  pathway 
leading to the deamination of L-ser ine .  
The distinctive spec t r a l  p rope r t i e s  of this 
We 
It  appea r s  


Structure and function of proteins and nucleic acid Progress report, 1 Jul. - 31 Dec. 1967

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