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99

DYNAMICS UP GENETICALLY-ENGINEERED ENZYMES: FLUORESCENCE AND

DEBOLARIZIID RAYLEIGH SCATTERKNG STUDIES QP YEAST' PHCISPHOGLYCERATK

KINASE

ALAN COOPERl, 'BETH SANDERS1 and DAVID T-F- DRYDENZ

IDepartment of ChemTstry, Glasgow Univerei.ty. Glasgow G12 8QQ. Scotland. U.R,

ZDepartment of Biochemfstry and Genetics, University of Newcastle upon Tyne. Newcastle NE2 4Hl-l. U.K.

SUHMARY Mgand-induced changes in low-frequency motions in lysozynle

(IX 3.3,L.LTJ ad gn botlj the urS1d-Q_vpa and H+~~BJX--_)G~ mu+Laf. f<tJrms 0% gM5Bk 3-phcs$l~o&~c@5rrbte kkDl3230 )Ex/ ac 2s3_2,3) have heren atuBSeb b. depo1arLzcfZi Rayleigb scattering in the D-3DD cs~-l rranga. Bfn#fng 0% linh1~1t~rs to 1y~ozys~e px&h~es changes %n the RayleSgh profIle conaiatant ~5th nn overall "atLf%en5ng" of the pXXke'rR\ Stm't1ar afeectff UC@! seen upoci btrr~lng of e&%rtrutet3 CXlX? OF ATP) to the cmcleatkde bfndfng &t&rak,a <IL mutant PGK but, kn

contrast, add5t5on of phosphoglycerate to the b-lLOEC3 bInding doma5n seems to result in an overall "loosening" of the protein structtrre. No such effects are observed rfth wild-type PGK. Doth etatfc and picosecond dynamic fluorescence studies of PGK ShOU

significant differences between the wild-type and mutant enzymesI rith evidence for an increase in tyrosine-tryptophan energy transfer in the hinge-bending mutant, Paradoxically, neither the eteady-state nor dynamic fluorescence properties appear to. he affected by substrate binding, possibly because of the different time scales involved for ligand-induced changes-

INTRDDUCTIUN

The dynamLc nature of the conformations of biological macro-

molecules is now well established both by experimental

cobs~rvation anZi *v 'theoret+ca'l preb~c?~o~ >reSs, '1-53, ax& rnnp bx~

viewed as an inevitable consequence of thermal fluctuat5ons in

meaoscopic systems. What is less clear-is the extent to which

proks'lns, tar 1RbtuRCe, have svuive& to take spe&P'Ic Iunck'lonai

&~uantnge a,f thsse inhers3nt f1uctuatLone, Clearly thar~ ar+a nrany

0167-7822/89/$03.69 6 1989ElsevisrB~iencePublishernB.V_

biomolecular processes which would be impossible, in the form we

see them today, in the absence of relativoly large conformational

fluctuatfons- For example: the binding of ligands or substrates

to apparently inaccessible buried sites, the bringing together of

different substrate molecules during catalytic events on multi-

substrate enzymes, the catalysis of reactions in the absence of

competing water molecules, long-range cooperat&vity and

alIosteric effects, and so forth, would not be possible but for

the dynamic flexibility of the protein structures Involved [refs.

4.6.71, We are familiar with the idea - though we do not know how

it is done - that the amino acid sequences of protesns have

evolved to al1ow folding of the polypeptide chain fnto the

(static) conformations required for their function. Is it not

also possible that these sequences have also been selected for

the dynamic possibilities that they allow? The availability of

routine site-directed mutagenesis techniques now allows us to

begin to test this possibility. Unfortunately there ls a shortage

of convenient experimental techniques for studying protein

dynamics, and those that are available span a wide range of time

scales- Consequently we have been exploring the possible use of

depolarized Rayleigh scattering (low--frequency Raman

spectroscopy) of dilute protein solutions to probe these motions

fn the thermally accessible region of the vibrational spectrum

(O-300 cm-l)- We -report here some preliminary observat5ona on

possible ligand-induced changes to the dynamic properties of

Iysozyme and of two forms of yeast phosphoglycerate kinase using

this technique, together with some comparative stud&es using

conventional fluorescence methods.

3-Phoaphoglycerate kinase (PGK; EC 2.7.2-3) is an enzyme of

the glycolytfc pathway responsible for the reversible synthesis

of ATP by phosphoryl transfer from 1.3-diphoephoglycerate <DPG)

to ADP. releas&ng 3-phosphoglycerate (3-PGA) ln the process- X-

ray crystallographic studies [refs- 8.91 have shown that it is a

monomer%c enzyme of 415 amino acids (mw 44,500) comprising two

well-defined structural domains connected by a narrow nec?c -or

"hinge" region, The binding sites for-the different triose and

nucleotide substrates are carried on these separate domains at

positions which, at least in the "open" conformation revualed by

crystallography, are too far apart for direct contact between the

substratus. It is postulated that during the catalytic event

.these two domains move closer together, probably by some "hinge-

101

bending" type of motfon. to bring the subetrate molecuIes into

JuxtaposTtTon and allow direct phosphate group tran6fer. This

would have the functional advantage of squeezfng natur molecules

out of thu active site region and preventing the occurrence of

competetlve hydrolysis rather than transfer of the phosphate

group. One key interaction in the hinge region of PGK which mPght

be implicated in-this process involves a salt bridge or e1ectro-

static gnteraction between histidine 388 and glutamate 190. Site-

directed muta-genesis techniques have been used to replace this

histidfne residue with glutamine [ref. 10) to produce a protein

with reduced specific activfty but similar substrate bindfng

properties to the wild-type enzyme. Both form8 of the enzyme are

studied here.

MATERIALS AND METHODS

Wild-type PGK wae prepared from drfed baker's yeast by

conventfonal technique8 combining ammon5.a lysis. ammonium

sulphate fractionation and column chromatographic procedures

[ref. 111. The His388-- >Gln mutant was isolated from an over-

expressing yeast strain containing multiple copies of the

appropriate plasmid- Yeast cells transformed using the pMA4Ob

plasmfd containing the modified yeast PGK gene (a gift from Dr-

L.A. Gilmore) were cultured in 10 litre batches and, after

harvesting, were treated aa above to yield the mutant enzyme. In

more recent work we havu used over-expressrng wild-type yeaet

mutants (pMA27 plasmid) to produce sample8 of unmodified enzyme

with no apparent change in results. After purification. PGKs were

stored in the cold as ammonium eulphate suapen6ions until

required. Enzymes purified in this faehion give intense single

bands of appropriate molecular weight on SDS-polyacrylamide

electrophoresis and chow enzyme kinetic parameter8 (Kn,vnrrr)

comparable to literature values,

Hen egg white lysozyme (EC 3.2,1_17). N-acetyl-D-glucosamine

(NAG). tri-N-acetylglucosamine (tri-NAG.tri-N-acetylchitotriose),

adenoaine 5' -diphosphate (ADP), adenosine 5' -triphosphate (ATP),

3-phosphogIyceric acid (3-PGA) and buffer salts were purchased

from Sigma. Lyaozyme sample8 for RayleigQ scattering studies were

made up in O.lM acetate buffer. pH 5.0. and dialysed briefly

against the same buffer immediately before Use- PGK r3ample8’ for

Rayleigh scattering were dispereed in. and dfalysed extene5vely

against 1OmM Tris/MOPS buffer, pH 7-O. with 4mM MgClz and O.lmH

dithiothreftol. Samples for fluorescence studies were dfssolved

in 10&l Tris, 9OmM NaCl. 1mM NazEDTA buffer at pH 7-27. wfth the

addition of 2.5mM MgCl z when nucleotides were present. To remove

aggregated material, all protein solutions were centrrfuged

extunaively and passed through 0.22~ Millipore filters before

use. Protein concentrations were estimated from the 2aonm

absorbance using extinction coefficients of 2-65 mg-lml cm-l and

0.495 mg-lml cm-1 for Lyaozyme and PGK. respectively.

Depolarized Rayleigh Scattering

Sample solutfons in lcm cuvettes 'were irradiated with

vertically-polarized 488nm light from a Spectra-PhysLcs Model 171

argon ion laser with intensity, measured at the sample, of 50 mW

OX- less. using narrow-band interference filters to eliminate

plasma lines. Horizontally-polarized (depolarszed) 90° scattered

light in the -100 to 300 cm-1 range was analyeed using a Coderg

triple-monochromator Raman spectrometer, with cooled

photomultiplier and computer-interfaced photon-counting

electronics, fitted with a Polaroid analyzer and polarizat%on

scrambler at the entrance slit. The spectrometer slfta WOK-t?

usually set at 70 microns throughout, corresponding to a spectral

bandwidth of about 0.8 cm-1 under these conditions. Spectra were

collected at a scan rate of 5 cm-lmrn-1 with a 5 or 10 second

count gate. To avo%d photomultiplier tube saturation, calibrated

neutral density filters were inserted in the collection optics

whslst scanning through the intense Rayleigh line (from -10 to

+lO cm-l). The linearity of the PM tube response under these

conditions wa6 also verified using neutral density fil.ters_ In

some experiments, designed to study the effects of ltgand

bind-lng. the spectrometer was scanned manually and sample8

interchanged at each fixed wavenumber position to eliminate any

possible scanning artefacte.

Fluorescence Studies

Steady-state fluorescence emission spectra (S-1Onm band-

width) were measured at 25oc using a Perkin-Elmer MPFJ-L

spectrofluorimeter at a variety of excitat5on wavelengths in the

250-29Snm region (3nm bandwidth), using semi-micro cuvettes and a

sample absorbance of less than 0.2 at the excitation wavelength.

Unpolarized protain fluorescence decay kinetics in the p,icoeecond

range were studied using an Edinburgh Tnatrumenta Model 199 T-

103

geometry spectrofluorometer with a nanosecond pulse flaahlamp

operated at 50 kRz which. after deconvolution. can resolve major

components in a multi-exponential decay down to about 300

picoaeconde. Excitation and emission wavelengths were normally

eet to 295nm and 35OIIllI. with 1Onm and 2onm bandwidths.

respectively, and data collection for each sample was continued

until at least 10,000 counts accumulated in the peak decay

channel. The lamp pulse profile under the same conditfons was

determined after each measurement using a standard Ludox

scattering sample. Multi-exponential decay data were analyzed for

best lifetime/amplitude combination6 by non-linear least-squares

deconvolution techniques using reduced chi-squared tests and

residual6 analyein as fitting criteria [ref. 123.

RESULTS

Depolarized Rayleigh Scattering

Aqueous protein eolutions show intense depolarized Rayleigh

scattering wfth approximately Lorentzian profile6 devoid of any

discernible vibrational structure (Pigs_ 1 and 2). At the

concentrat5ons used routinely here (x2-8%). this scattering 56 at

least two orders of magnitude greater than the solvent background

(buffer or buffer plue ligande) and is linear with protein

concentration over at least a lOO-fold dflution- Despite the lack

of fine structure in these spectra, significant change6 were

observed upon ligand bfnding, in some cases, With lysozyme, for

example. addition of the specific inhibitor tri-NAG produced a

broadening of the Rayle5gh peak together with a reduction 3n peak

hekght consistent with a shift %n vibrational population from

very low to higher frequencies (Fig-l). Similar effects were Been

with the monomeric fnhibitor. NAG (O.lZM), but not in control

experIment6 involving addition of simflar concentration6 of

glucose, which doe6 not bind to the enzyme-

The situation with PGK is somewhat less clear cut, Por

reason6 which are not yet clear to us, but wh-lch may be

aeaociated with a tendency for this protein to aggregate under

some conditions, scattercng from PGK solutions was frequently

noisy and erratic, making observations d%ffXcult. Within these

constraints the wild-type enzyme showed no consistent change6 *n

d-NAG 0

cm -1

Fig. 1. Depolarized Rayleigh scattering spectra of 5mM in O.lH Na-acetate buffer, pH 5.0.

lysozyme in the presence and absence of

5.5mM tri-NAG. The insert shows the differences in peak height on a different scale,

-ATP/s-f’G

- l-PG

‘-‘T .*A.. - PGH

b

-ADP

+lmM ADP -4 0 4

em a-s.---...r_ - -u--r

.a

+l mN I 3-PC

I , I ___

-100 0 100 200 309

cm -1

Fig. 2, Depolarized Rayleigh scattering profiles of mutant PGK and the effects of substrate bindfng (3-PG: 3-phosphoglycerate).

scattering profile on addttion of any of the substrates. In

contrast, however, the His388-- >Gln mutant enzyme did give marked

changes, and in opposite directions (Pig.2). Binding of ADP and,

to a lesser extent, of ATP to mutant PGK resulted in an intensity

decrease and broadening of the Rayleigh profile qualitatively

similar to that observed for lysozyme. Addition of 3-PGA.

howwvar. produced the opposite effect with a distinct narrowing

of the Rayleigh band coupled with an increase ln tha peak height.

as did the combined addition of equal concentration9 of both

substrates. ATP and 3-PGA. which under these conditfons would

immed5otely undergo enzyme-catalyzed formatgon of the equilibrium

product mixture of ADP and 1,3-diphosphoglycerate_ Bearing In

mind that the nuclwotide and triose binding sites occur on

different domains of th5s enzyme it seems clear, in the caew of

this modified protein at least, that ligand binding has a

charactaristically different effect in each domain and that, with

ternary substrate complexes. the effects on the triosw binding

domain appear to predominate.

Fluorescence Studies

Signif%cant differences were observed in both steady-state

and dynamic fluorescence properties of wild-type and mutant PGKs.

The fluorescence emfesion spectrum of wild-type PGK 5s known to

be untypical [ref- 131 since it shows a dominant tyrosXnw

emission (Xs~~31Onm) at excitatlion wavelengths below 290nm where,

in most prote5ns containing both tyrosine and tryptophan

residues, energy transfer gives3 rise to more tryptophan-like

emission spectra (XEna33Onm). This is evident in Table 1 from the

distinct shift in Xsn with excitatgon wavelength at around 290nm.

By contrast, the behaviour of the mutant enzyme is more normal in

this respect and shows only a small variation in )ren w5th %5X*

with an emission profile characteristic of pure tryptophan

throughout the excitation spectrum indicative of more efficient

tyr-->trp energy transfer in the mutant as compared to the wild-

type. Despite these differences. however. neither the mutant

(Table 1) nor the wild-type enzyme (data not shown) show any

significant variation in excitation or emission spectra when

substrates are bound.

Significant differences between the two forma of the enzyme

are also apparent in the picosecond fluorescence decay data

(Table 2). Both forms show the unambiguous three--exponential

tryptophan decay CharacterLatLc of this enzyme Crefa. 14.151 with

quite similar relaxation times, apart from a possibly slightly

faeter slow component for the mutant PGK. However, there are

signrficant reproducible differences in the amplitudes of the

three kinetic components, with the mutant showing a distribution

shift from the dominant 330 paec relaxation of the wfld-type

towards the slower (-2 and 5 nsec) components. But, again, none

of these properties appear to be influenced by binding at either

of the substrate btnding sites.

TABLE 1

Steady-state fluoreecence emission maxima of wild-type and mutant

<Hifi388-- >Gln) PGK.

Excitation Emission Maximum (rt2 nm) Wavelength <nm) Wild Mutant Mutant Mutant

Type + ATP + 3-PGA

250 311 322 -

260 310 322 270 310 320 280 306 321 321 321 290 320 326 -

295 329 320 327 330

TABLE 2

Fluorescence lifetime and amplitude parameters for the triple-

exponsntial decay of wild-type and mutant PGK (295nm excitation*,

standard deviations in parentheses).

Lifetimes (7) Wild Mutant Mutant Mutant Amplitudes (a) Type + ATP + 3-PGA

71 n8ec. 0.38 (-02) 0.33 (-02) 0.29 (-02) 0.33 (-03) 71 nBec. 2.23 (-06) 2.13 (.07) 2.02 C-09) 2.43 C-07) Tzi nsec. 5.45 (-09) 4.72 C-04) 4.68 (-04) 5.33 C-06) a1 Z 62.0 35.8 36.1 35.8 a1 % 26.0 31.3 28.4 38.8 o[s Z 12.0 33.0 35.5 25.4 ChiZ 1.11 0.99 1.03 0.98

"similar results obtained with 280nm excitation.

107

DISCUSSION

Model normal mode calculations on globular proteins [ref.

I61 show a wealth of vibrational modes below about 300 cm-1

involvilrg large scale cooperative motione. including "hinge-

bending- type motions between structural domains and involving

active sites. which should ba thermally excited under

physiological conditions. But. apart from a few Xnstances with

crystalline or dry protein samples [refa, 17.181. low-frequency

Raman or other vibrational spectroscopic techniques have failed

to detect such modes in real proteins. The problem seems to be

that. as a result of the inevitable anharmonicity of the

intramolecular potentials and of the viscous damping effects

(both internal protein and external solvent effects), these

harmonic modes collapse into very low frequency, non-harmonic.

stochastic, diffusion-like motions wh%ch will appear only as an

addttional broad contribution to the -Rayleigh scattering peak of

the Raman spectrum. Because of thu anisotropic nature of the

motions, however, this contribution should have a strongly

depolarized component which might allow it to be distinguished

from the normal intense, polarized, non-speciffc Rayleigh band.

Depolarized Rayleigh acattersng has been used extanaively in

recent yeara to study the dynamics of simple lfquids [ref. I91

but has not, to our knowledge, hitherto been applied to protein

solutions. The preliminary results reported here, which show that

dilute solutions of proteins under nearly physiological

conditions do exhibit a strong depolarized Raylefgh band which

can be influenced by substrate and inhibitor binding, indicate

that this technique is of some promise. (Rut. a word of cautfon

before we proceed: the theoretical basis of depolarized Rayle5gh

scattering from macromolecules such as these has yet to be

established. and we have yet to prove that the changes that we

observe are a direct dynamic consequence of ligand binding and

not due to some indirect effect such as ligand-snduced

aggregation effects, or whatever.)

Despite th5e caveat, intuitfon supported hy molecular

dynamics simulation8 [ref. 201 and thermodynamic data [refa.

3,4,211 suggests that binding of any ligand to a protein active

site, especially if that site lies in a cleft between structural

domarns, should make the macromolecule more rigid or "stgffer"

and should shift the dynamic frequency distribution of the

protein to higher frequencies- The observations on lyaozyme seem

to bear th%s out since brnding of NAG or tri-NAG (Fig.lj in the

active site cleft results in a redistribution of scattering

intensity from very low frequency to h5gher (but SIC511 -low* 5.n

conventional terms) frequencies in both the Stokes and anti-

Stokes (thermally populatud) regions of the depolarized Rayleigh

band,

Substantially similar stiffening effects are observed upon

binding of substrate molecules to the nucleotsde binding site of

mutant PGK. But precisely the opposite effect is seen upon

binding of 3-PGA at the opposing triose binding site. as shown by

the transfer of scattering intensity from the higher frequency

region to the very low frequency Rayleigh peak (Pig.2). This is

unexpected but leads to an intriguing picture of how thfs enzyme

might actually operate. Recall the conventional view in which the

bsndlng of substrate molecules to PGK forces a conformat*onal

change in the protein that draws together the active site

regions, evicts water and allow6 the catalytic act to proceed.

Following this a second conformational change must occur to allow

release of the products. (There is apparently a paradox here: the

model postulates that the ternary PGK-ATP-PGA complex must be in

a "closed" conformation and, following phosphate transfer. the

product complex PGK-ADP-DPG must "open" to release the products.

But PGK. like all enzymes, works equally well In both directions

and, indeed. does operate in the direction of ATP synthesis

during glycolysis. Hsnca the PGK-ADP-DPG complex should be

"cloeed".)The alternative picture suggested by the results here

5s that the "hinge" in PGK is perhaps rather stiff and that

binding of 3-PGA, rather than forcing a concerted conformational

change, simply "lubricates" or "unlocks" the hinge to give a more

flexible molecule In which the substrate domains may non approach

each other in a much more random fashion under the influence of

thermal fluctuations in a diffusive, Drownian-mot5on-like manner,

The dsfference is subtle, but this dynamic mechanism has the

advantage that no independent second process need be postulated

to facilitate subsequent product release, and the enzyme works

equally well in either direction. Since the hinge rema5ns

unlocked after the catalytic reaction, as indicated by the

ternary PGK-ATP-PGA complex data (Fig.2). product release can

take place by the same stochastic hinge-bending motion because

only when the triose product is released does the hinge seize up

agaln.

109

Our failure to detect comparable effect6 with nfld-type PGR

is gutzilng, bu* cone must aiway6 bc alure t'nat even aingie amino

acich anY&r5%V~!1on~~ rn?&Wc 'nave long ra~l,a~ e%fecta on proteTn

conformation or stability in addition to the changes one is

att~n&in~ tr> ursg5xMStr, an0 it 'is ;poaai~Ia that tha &ynnS&c

effects have been ampl5fied in this mutant by the weakening of

the interactions in the hinge region or by more inilrect glabal

effects arieing from the mutagenesis.

The possibility that the hietldine388->glutamfne mutation

has introduced long range changes in the static and dynamic

structure of PGK ie supported by the fluorescence data. Although

the fluorescence relaxation times are similar for the mutant and

wild-type enzyme, there is a considerable difference in the

dfstribution of relative amplitudes of the components between the

two. Even 030~43 etriking are the differences in steady-state

excitation/smisaion spectra, where the relative red-shift of the

emission maximum in the case of the mutant protein at short

wavelength excitation is suggestive of some increase in

fluorescunce energy transfer from tyrosine ta Lryptophan compared

to the wild-type enzyme, All this indfcatee that the static and

dynamic env5ronmunta of at least some of the aromatic residues in

PGR have been altered by mutation of His388 go a glutamine

rcm%&ne, TG;k cun*%a'lna 'tu-0 kmio$nan a>be c'na'lna ~"SiSb ai% "s15:1,

both located in the nucleotide binding domain but remote from the

active Esite and from tha situ of mutagenesis [ref. 91, In

adcb5tXon 'chore aru ae-ven 'tgros'l_Tle reaL&uea, aI8Tz r=rnt?c.% fxmrnt!=

ac*fv9 sI!ze rq@ozrs tit &bh orre, TJY&X+, >ocab& 522 *??a- ?&!?grr

region close to Glul90 which interacts with the His388 residue

subject to mutation here. Tyr1.93 is eeeential for PGK activity

and Beem to be 5nuoIved 5-n the hinge bend'ing meehnni8m srBiB_

9,221. It seems possible, therefore, that mutation of His388 has

altered the conformation of the protein in the region of Tyr193.

and possibly elsewhere. so as to affect the energy transfer and

relaxation properties.

But, in view of this. it is surprising that in no case is

the fluorescence affcctud by substrate binding, The argument goes

aa follows: if Tyr193 5s involved in the hinge mechanism and if

its conformational state affects tryptophan fluorescence, then

substrate binding (which we believe affects the hinge) should

indirectly alter the Trp fluorescence properties. There are at

1aast tpro po?xafbZe explanatLons of this paradox: either the

110

ligand-induced dynamic changea we observe by Rayleigh scattering

are not associated with changes at the hinga, or tha time scales

involved are too disparate for the antic3pated effects to occur-

Bear in mind that the typical fluctuation time associated with

the Rayleigh scattering measurements (l-380 cm-r, say) is of the

order of 0.1 to 30 psec, compared to 300 psec or more #or the

fluorescence lifetimes. Thus the functionally related dynamic

fluctuatfons ln the hinge region of the protein may be too fast

for their effects to be detectad by fluorescence methods, even

though they are observable by depolarized Rayleigh scattering.

ACKNOWLEDGEMENTS We would like to thank Dr. L.A. Gilmore for the gift of

plasmids. Dr. A. Brown for help wfth yeast transformations. Prof. L-D. Barron for use of the Raman facilities. and the SERC for financial support-

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