The to ta l energies of th e sp ecies discussed below are given in Table 6-1. An i n i t i a l d iscu ssio n of the o v e ra ll re a c tio n scheme i s given here.
The th io l-s u b s titu te d model scheme - th a t i s th e o rig in a l scheme - does not fe a tu re such la rg e energy d ifferen c es between th e
in term ed iates as th e hydroxy- and h y d rogen-substituted schemes. The o v e ra ll re a c tio n shows a s ta b ilis a tio n of -0,0138 a .u ., compared w ith -O.O327 a .u . fo r th e hydro x y -su b stituted scheme and 0.0 a .u . fo r th e hydrogen-substituted scheme, in which A i s the same molecule as H and C i s id e n tic a l to F. Thus, in terms of the o v e ra ll re a c tio n , i t would p ro f it the enzyme to use an oxygen-based s u b stra te in stead of a sulphur-based one, as th is i s th e most e n e rg e tic a lly favourable re a c tio n . However, i f one looks a t the energy d iffe re n c e s along the proposed pathway, i t i s obvious th a t th e re is a s u b sta n tia l gain to be achieved in using sulphur, in th a t the a c tiv a tio n en erg ies needed to remove th e protons from th e molecule are le s s severe in the case of the th io l-s u b s titu te d A->C and E->F re a c tio n s than in e ith e r of the two analogous schemes.
The choice of is o la te d m olecules, n eg lectin g solvent e ffe c ts , is ju s tif ie d because th is p a rtic u la r enzyme must have a very w ell
p ro tected a c tiv e s ite . This i s shown by the d if f ic u lty in observing solvent proto n atio n when th e re a c tio n was c a rrie d out in D^O [73,74] or tritiu m -e n ric h ed w ater [75], which led to the b e lie f th a t the enzyme cataly sed a re a c tio n analogous to th e Cannizzaro re a c tio n . The proton tra n s fe r i s so w ell shielded th a t the solvent does not play a g reat p a rt in th e re a c tio n , as shown by th e work of H all e t a l. [78,79],
where only a sm all amount of so lv en t in co rp o ratio n was d etected by nmr.
Thus we do not s u ffe r from th e g re a t bugbear of b io lo g ic a l c a lc u la tio n s : so lv atio n e f f e c ts . This to p ic has an undeserved re p u ta tio n as th e stic k in g p o in t of mar^ th e o re tic a l sim u latio ns of chemical re a c tio n s; th is does not apply exactly to th e co n sid eratio n of enzyme s ite s , as these o ften have highly p o larised regions not sim ila r to a wet chem istry environment, and the only way to tr e a t th is
s itu a tio n , in th e absence of d e ta ile d inform ation of the a c tiv e s ite s tru c tu re , is a c a re fu l exam ination of the p ro p e rtie s of the v ario u s su b stra te s and in h ib ito r s . This i s attem pted in the follow ing
ch ap ters.
6.3 The S u b strate Model: Molecule A
This p a rtic u la r sp ecies i s a model fo r th e glutathione-aldehyde adduct, the h em ith io acetal used by glyoxalase I , The chemical groups not joined to th e oxygen atoms have been replaced by hydrogen because of computer disk space lim ita tio n s . This enabled the u tilis a tio n of a s p lit-v a le n c e b a sis s e t w ith d -o rb ita ls on th e sulphur atom as
discussed above. The groups replaced by a hydrogen atom were the a lk y l or phenyl group of th e aldehyde and th e g lu tath io n e trip e p tid e , except fo r a re sid u a l th io l group.
Molecule A has been stu died a t both the 3-21G and 3-21G* le v e l, in order to examine th e e ffe c t of the d -o rb ita ls of the sulphur upon the bonding and chemical p ro p e rtie s. F u ll geometry o p tim isatio n s, using both these b asis s e ts have been made. The re s u ltin g geom etries are
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lis te d in Table 6-2 and th e 3-21G* geometry is shown in Figure 6-2. The la rg e s t d ifferen c e between the two geom etries i s a 4%
shortening of th e C-S bond len g th upon the a d d itio n of d -o rb ita ls to the sulphur b a sis s e t. This immediately suggests th a t th e re i s some (d-p) Tf bonding between th ese two atoms. Other larg e e ffe c ts are the widening of the 03-C1-C2-H7 d ih ed ral angle (2.7%) and th e 03-C1-C2-S5 angle (1.0%), such th a t th e sulphur approaches the 03-C1-C2 plane, pushing th e hydrogen fu rth e r away from i t . The sulphur hydrogen bond length i s also decreased by 1.8%. The C1-C2-S5 bond angle is increased by 1.6% and th e C1-C2-H7 bond angle i s decreased by th e same amount. The r e s t of th e molecule is a ffe c te d by le s s than 1% changes. Thus, as would be a n tic ip a te d , th e geom etrical param eters in th e region of th e
sulphur atom are th e only ones to be influenced by the ad d itio n of the d -o r b ita ls .
The main e n erg etic e ffe c t of adding d -o rb ita ls to the sulphur atom is a low ering of th e to ta l energy of the sp ecies, which i s to be
expected from a purely v a ria tio n a l point of view, as th e a d d itio n of e x tra b a sis fu n ctio n s allow s more f le x ib ili t y to the wave fu n ctio n d e sc rip tio n .
The la rg e s t percentage change in an o rb ita l energy i s th a t of o rb ita l 23, by 2.3%. The d -o rb ita l co n trib u tio n i s not of s-c h a ra c te r in th is case: th e la rg e s t c o e ffic ie n t i s th a t of th e d -fu n c tio n , w ithxy a s lig h tly sm aller d yy c o n trib u tio n . The d - and d -fu n c tio n s haveXX zz n e g lig ib le c o e ffic ie n ts . As th e re are also la rg e exponents on the 2py-functions on C2, i t seems lik e ly th a t the C-S bonding d isp lay s some (d-p)IT type in te ra c tio n .
The e ffe c t of th e a d d itio n a l fu n ctio n s on the sulphur can also be noted in the charge d is trib u tio n fig u re s from a M ulliken population a n a ly sis. Considering th e to ta l atomic charges f i r s t , th e re i s l i t t l e or no d ifferen c e in th e charges on Cl, 03, H4, 06, H7 and H9, but the d ifferen c e in charge along th e C2-S5 bond i s reduced considerably, as th e charges on C2 and 85 a re reduced from -0.23 to -0.16 and 0.05 to -0.01 re sp e c tiv e ly . The overlap between C2 and 85 shows a s lig h t
in crease in p opulation, as does the sulphur atom occupancy. The number of e le c tro n s asso ciated w ith C2 d ecreases. Thus, th e basic trend in th is reg io n , upon ad d itio n of d -fu n ctio n s, i s th a t fewer e le c tro n s are a sso ciated w ith C2 near i t s nucleus and more are asso ciated w ith 85. The net e ffe c t i s to reduce th e negative charge on C2. This property of the sulphur atom may be thought of as helping to s ta b ilis e any anion formed by the removal of th e proton, H7. This point w ill be discussed more fu lly l a t e r on.
A fu rth e r c a lc u la tio n was made using the 6-31G** b a sis s e t , w ith th e 3 -21G* optim ised geometry. This was made p o ssib le by an a llo c a tio n of resources on th e CYBER 205 a t M anchester. Time lim its prevented a complete geometry o p tim isatio n , but the improved b a sis s e t should shed fu rth e r lig h t on th e e le c tro n ic s tru c tu re of th e m olecule, a lb e it a t th e 3-21G* geometry.
The ad d itio n of d -fu n ctio n s to the carbons and oxygens and
p -fu n ction s to the hydrogens re s u lte d in a low ering of the to ta l energy of the species by 3.133 a .u . The wave fu n ctio n was sim ila r to the 3-21G* one, showing s ig n ific a n t p a rtic ip a tio n of the d -fu n ctio n s of sulphur in some of th e bonding o r b ita ls . O rb ital 23 of the wave function in th is b a sis also shows a s ig n ific a n t c o n trib u tio n from the d -fu n ctio n on th e sulphur and th e p -fu n ctio n of 02. The to ta l amount
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of e le c tro n p a rtic ip a tio n in th e d -fu n ctio n s on th e sulphur fo r th is b a sis s e t i s 0.094 e le c tro n s as opposed to 0.127 e le c tro n s w ith th e 3-21G* b a sis s e t. In f a c t, th e to ta l fo r Cl, of 0.093, and C2, of 0.095, are sim ila r to th a t of the sulphur.
The su b stra te model was fu rth e r in v e stig ate d by th e use of two s u b s titu tio n s : the replacem ent of th e th io n y l group by a hydroxy group and by a proton, c re a tin g two new molecules in a d d itio n to A. The optim ised geom etries of th ese th re e m olecules d if f e r markedly: upon rep lacin g the sulphur, th e carbon-carbon bond i s shortened in both cases.
The main change in the s u b stra te model upon rep lacin g th e th io l group with a hydroxy group, a p art from the obvious change in charge on X5, i s the a lte ra tio n of the charge on carbon C2 from -0.16 to 0.34. The more e lec tro n e g a tiv e oxygen has drained the carbon of some
e le c tro n ic charge, making i t more p o s itiv e . This can be seen in the overlap population of th ese two sp e c ie s: th e population centred a t C2 i s 5.53 and 5.01 re sp e c tiv e ly fo r molecule A and the
hydrogen-substituted molecule A. The r e s t of th e molecule is comparable to th e su b stitu te d v ersio n , except th a t th e C2-G6 bond overlap has changed from 0.42 to 0.47.
The h y drogen-substituted compound has very sim ila r charges on the atoms to the th io n y l compound, except th a t the protons H4 and H7 are
s lig h tly le s s p o s itiv e . H7 i s a lso le s s p o sitiv e in the
h y droxy-substituted m olecule. I f we look a t the overlap population in th is case, we fin d th a t th e re i s a d ifferen c e in th e overlap on C2, as before, which i s reduced from 5.53 to 5.31. Also th e C2-06 bond
The bond overlap of H7 to C2 i s constant fo r a l l th re e m olecules, being 0.70, and th e overlap centred on H7 i t s e l f r is e s from 0.51 fo r molecule A to 0.53 fo r th e hydrogen su b stitu e n t and then to 0.58 fo r the hydroxy m olecule. Thus, i t does not seem, (e sp e c ia lly i f we a lso consider th e bond d ista n ce d a ta ), as i f th e enzymic re a c tio n gains any advantage from a weakening of the aldehydic proton binding - i f
anything th is i s strengthened. However th e e n erg etic s ta b ilis a tio n of molecule C w ith refe re n ce to molecule A seems a ju s tif ic a tio n fo r th e use of th e h em ith io acetal as an i n i t i a l s u b stra te . This c h a ra c te ris tic does indeed seem to be a re la tiv e s ta b ilis a tio n of C, ra th e r than a d e s ta b ilis a tio n of A, as th e aldehydic proton does seem to be more secu rely bound to C2 in th e su lp h ur-con tain ing molecule A than in the o th er two cases, so th e removal of the proton would be more d if f ic u l t. This, then, is compensated fo r by the increased s ta b i lity of th e created anion, molecule C.
I t appears th a t the d -fu n ctio n s do allow the molecule to indulge in some d-p in te ra c tio n , but th a t i t i s not as im portant in d efin ing th e chem istry of th is compound as was o rig in a lly thought. I t would be in te re s tin g to attem pt a geometry o p tim isatio n of th is molecule (and o th ers in th is model scheme) a t th e 6-31G** le v e l, as recommended by Wolfe e t a l. [158], to see i f th is made much d ifferen c e to the
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6.4 The Anionic Hpeoles.
There are fiv e anionic m olecules in th is model scheme: four w ith a sin g le , o v e ra ll, negative charge and one dianion: