FUNGAL CELLULASES by M. A. Jermynt Those Glycosidases That

------

FUNGAL CELLULASES

XV.* ACCEPTOR SPECIFICITY OF THE ARYL ,B-GLUCOSIDASE OF STACHYBOTRYS ATRA

By M. A. JERMYNt

[Manu8cript received March 31, 1966]

SU'YYIIJnary A parameter has been devised that gives a general measure of acceptor efficiency in the reaction of an enzyme with two substrates. The numerical value of this parameter has been determined for the ,B-glucosidase of Stachybotrys atm, phenyl ,B-D-glucopyranoside, and a large number of hydroxylic acceptors. There are certain correlations between acceptor efficiency and structure.

1. INTRODUCTION Those glycosidases that transfer the glycosyl residue to an acceptor with retention of the configuration about the anomeric carbon atom may be taken as "typical". Although "atypical" glycosidases which give rise to products that have inverted configurations or are unsaturated may share with the typical enzymes many common elements in mechanism, not enough is yet known about the nature and function of active centres in either class to make this more than a speculation. The observations recorded in this and the following papers are therefore only directly relevant to the problem of the mechanism of typical glycosidases. The latter enzymes and those of the protease-esterase group both catalyse (for hydroxylic acceptors) reactions of the type

XOR+R'OH ~ XOR' +ROH. The crucial difference in mechanism appears to be that the protease-esterase reaction proceeds in two steps, enzymeOH + XOR -+ enzyme OX + ROH enzymeOX+R'OH -+ enzymeOH+XOR' but that formation of enzymeOGly by glycosidases appears to occur as only a transient, perhaps as only a virtual, stage that is part of a single-step mechanism. This mechanism involves the decomposition of a ternary complex in which GlyOR and R'OH are simultaneously bound to the enzyme. No mechanism has yet been postulated, however, for glycosidase action that does not include such a virtual intermediate, implied as it is by the necessity of a double Walden inversion to restore the configuration about the anomeric carbon.

* Part XIV, AU8t. J. Biol. Sci., 1966, 19, 7l5~17. t Division of Protein Chemistry, CSIRO Wool Research Laboratories, Parkville, Vic.

AU8t. J. Biol. Sci., 1966, 19, 903-17 904 M. A. JERMYN

The best-known and most easily purified proteases have been studied exhaus tively. The sequence of residues in their peptide chains and much of the geography and chemistry of their active centres are known. The advantage in clarity that this gives can be seen by comparing such relatively sophisticated expositions of mechanisms as that of Bender and Kezdy (1964) for chymotrypsin and that of Ebert and Stricker (1964) for dextransucrase. In the first case defined bonds can be made to definite residues; in the second case complex thermodynamic data obtained by ingenious experiments must still be interpreted by full or broken lines pointing towards conveniently spaced functional groups on the surface of an undefined enzymic "plum pudding". For protease-esterases, direct evidence for the postulated mechanism is therefore possible in favourable circumstances, and a number of acyl-enzyme intermediates have in fact been isolated and the nature and position of the acylated group determined. For glycosidases the evidence is all indirect, in the sense that observations have been made that are incompatible with the two-step hypothesis. Thus the nature of the glycoside may determine the point of substitution in a polyhydroxylic acceptor (Miwa et al. 1956), or the ratio of the products when two competing acceptors are present (Jermyn 1962b). The idea of a ternary complex is itself susceptible to further analysis, and Ebert and Stricker (1964) have applied such an analysis to dextransucrase. Here, the highly anomalous kinetics of a complex situation with several competing processes involving a number of molecular species, each of which can serve as both donor and acceptor, can be simply interpreted in terms of the rates at which various bonds are formed and broken in a ternary complex. Glycosidases t,hus seem to be only a I:lpecial case of the class of "two-substrate" enzymes. The glycoside is only one of the substrates, certainly not the substrate; the acceptor is the other. Although much work has been carried out on the specificity of the reaction between enzymes and glycosides, little is known, on the other hand, about the specificity of the reaction between enzymes and acceptors. This little has usually taken the form of measurement of the relative transfer to water and acceptor at one or two concentrations of a few acceptors under fixed conditions. The ,B-glucosidase of Stachybotrys atra is very suitable for a more detailed analysis of acceptor specificity since certain complicating factors such as transfer of glucosyl residue to substrate glucoside, or product glucose and glucoside, or the hydrolysis of the product glucoside are absent or quantitatively negligible. An additional reason for the study was the suspicion that the hydrolytic action of the enzyme may not be a true indication of the role it plays in the integrated metabolism of the mould. The enzymic synthesis of a disaccharide by the transfer of a glycosyl residue to a glycose and the enzymic hydrolysis of a disaccharide are formally identical. Since the latter process is frequently highly specific for both moieties of the disaccharide, a sugar appeared to be the most likely candidate for the role of "true acceptor" of the ,B-glucosidase. The site at the active centre that binds the acceptor would then be expected to display a "true" specificity as narrow as that for the donor (Jermyn 1955b). The observed functioning of water and alcohols as acceptors would be no more relevant that the ability of all sorts of

[GluOR] [GluOR]

and and

[GluOH] [GluOH]

1O-

~ ~

-1O-

and and the the M, M, binding binding of of 5 these these 4 species species to to the the

enzyme enzyme

[PhOGlu] [PhOGlu]

10- ~ ~

and and M, M, a a degree degree

of of reaction reaction 3 not not exceeding exceeding 1-10%, 1-10%, the the resulting resulting

E . . E

PhOH. PhOH. ROGlu ROGlu

are are

excluded excluded from from

the the scheme. scheme. At At the the concentrations concentrations

used, used,

complex complex

(Cleland (Cleland

1963) 1963)

and and hence hence such such

transformations transformations

as as E. E. PhOGlu. PhOGlu. ROH ROH

~ ~

We We

cannot cannot

tell tell

from from kinetic kinetic

data data anything anything

about about events events within within the the Michaelis Michaelis

k_7 k_7 k_ll k_ll k-14 k-14

+PhOGlu +PhOGlu

~ ~

PhOH PhOH ~ ~ +E'GluOR +E'GluOR GluOR GluOR ~ ~

k7 k7 kll kll k'4 k'4

k_3 k_3

E+ROH E+ROH

~E.ROH ~E.ROH E. E. PhOGlu. PhOGlu. ROH ROH

k3 k3

k_6 k_6 k-ID k-ID

+ROH +ROH

~ ~ GluOR GluOR ~ ~

kG kG k'D k'D

k_2 k_2

13 13

E + + E PhOGlu PhOGlu

E. E. ~ ~ PhDGlu PhDGlu

+E.PhOH +E.PhOH

PhOH PhOH +E +E ~ ~

k2 k2

'3 '3 k

k_. k_. k_. k_.

2 +H

O O

~ ~ OluOH OluOH ~ ~

k. k. k. k.

k_1 k_1

'" '" E.H E+H 2 O O

O O 2

E . . E 2 PhOGlu. PhOGlu. H O O

k, k,

k_4 k_4 s s k_ k_12 k_12

+PhOGlu +PhOGlu

~ ~

PhOH PhOH ~ ~ +E. +E. GluOH GluOH GluOH GluOH ~ ~

k4 k4 ks ks k12 k12

ary ary 1 1 glucoside): glucoside):

covers covers

the the

various various

reactions reactions

taking taking place place (Jermyn (Jermyn

1962a) 1962a) may may be be written written (PhOGlu (PhOGlu

= =

presence presence

of of an an

inevitable inevitable

second second

competing competing

acceptor, acceptor, water. water. The The scheme scheme that that

Any Any

substance substance

that that

acts acts as as

an an

acceptor acceptor for for the the ,B-glucosidase ,B-glucosidase does does so so in in the the

II. II.

THEORETICAL THEORETICAL BACKGROUND BACKGROUND AND AND DEFINITIONS DEFINITIONS

S. S. atra atra into into a a new new and and more more satisfactory satisfactory picture. picture.

attempts attempts

to to

synthesize synthesize

what what

is is known known of of

the the mode mode of of action action of of the the ,B-glucosidase ,B-glucosidase

of of

terms terms of of

enzymic enzymic

mechanisms mechanisms

are are

now now seriously seriously out out of of date; date; this this series series of of

papers papers

therefore therefore been been

examined. examined.

Interpretations Interpretations

previously previously given given of of earlier earlier observations observations

in in

glucopyranosides glucopyranosides

of of which which

were were

wanted wanted

for for another another purpose purpose (Jermyn (Jermyn 1965), 1965),

has has

the the glucosyl glucosyl

residue residue

is is transferred. transferred.

Substitution Substitution in in the the glycitols, glycitols, the the mono-,B-D

can can

only only

be be assessed assessed

after after

the the identification identification

of of the the site(s) site(s) in in the the molecule molecule

to to

which which

most most

effective effective acceptors acceptors

are are

polyhydroxylic polyhydroxylic

and and quantitative quantitative data data on on their their behaviour behaviour

solutes, solutes, the the

behaviour behaviour

of of a a related related

non-hydroxylic non-hydroxylic

solute solute was was studied. studied. Many Many

of of the the

appear appear

to to show show

a a non-specific non-specific

effect effect on on

the the enzyme enzyme protein protein in in their their character character

as as

classes classes of of

acceptors acceptors

that that

could could

be be distinguished. distinguished. Since Since some some of of these these acceptors acceptors

details details

of of the the

investigation investigation

of of

the the

kinetics kinetics of of suitable suitable examples examples from from

the the various various

glucosidase glucosidase

of of

S. S.

atra atra for for a a

wide wide range range

of of acceptors; acceptors; the the following following papers papers outline outline

This This paper paper

records records

a a qualitative qualitative

investigation investigation into into the the specificity specificity of of the the ,8-

although although

this this

site site is is actually actually narrowly narrowly specific specific for for aryl aryl ,8-D-glucopyranosides. ,8-D-glucopyranosides.

hydroxylic hydroxylic

molecules molecules

at at

high high

enough enough concentrations concentrations

to to bind bind to to the the donor donor site, site,

FUNGAL FUNGAL CELLULASES. CELLULASES. XV XV 905 905 906 M. A. JERMYN

is negligible; in addition, all observations on the enzyme suggest that the affinity for PhOH is zero, so that all paths involving the species E. PhOH may be eliminated from discussion (Jermyn 1962a). The assumptions are, of course, far from valid for other glycosidases, but within their ambit the scheme reduces to:

k. +PhOGlu ;e= k_. kl ks k12 E+H.O ;e= E.H.O E.PhOGlu.H.O ~ PhOH+E.GluOH ->- GluOH k_l k5 +H.O ;e= k_5 k. E+PhOGlu ;e= E.PhOGlu k_. +E

k. +ROH ;e= k_6 k. kl1 k14 E+ROH ;e= E.ROH E.PhOGlu.ROH ~ PhOH+E.GluOR ~~ GluOR k_. k, +PhOGlu ;e= k_,

The mathematics of such a scheme have been developed elsewhcre (Jermyn IH()2a), but certain of its consequences may be deduced by qualitative reasoning. Let us define Tso as the concentration of added acceptor at which ,30(% of the glucosyl residue is transferred to this added acceptor and 50% to water. T7S and T 25 etc. follow obviously and the definition can be extended to any transferring enzyme. If t is the fraction of transfer to the added acceptor, then T50 is the con centration of added acceptor at which tl(l-t) = 1. It is apparent that two separate comparisons are confounded in T 50-the partition of E between E. GluOPh. ROH and E. GluOPh. H 20 in the steady state, and the relative rates of decomposition of the two complexes. Nevertheless it gives a measure of the "efficiency" of any given acceptor and no acceptor will be specific for which T50 is not very low. It is thus a useful parameter for a general survey of acceptors. In comparing adjacent members of homologous series it will give fairly close estimates of relative affinity; thus it will be justifiable to compare methanol, ethanoL and n-propanol, but not, say, methanol and sucrose. No useful meaning can be given to Tso unless the fraction of transfer, when the concentration of the added acceptor is fixed (that of water being fixed by circumstances) is independent of the concentration of the donor, [D]. Otherwise the fraction of transfer will be a constantly changing quantity during an experiment, as donor is used up, and will depend also on the initial concentration of donor. A different value of T50 will thus be deduced from the results of different experiments.

functioning functioning

in in

an an aqueous aqueous

environment, environment,

is is obviously obviously

fictitious. fictitious. in in such such T50 T50 cases cases

in in the the

last last example, example,

the the

calculation calculation

of of which which

moreover moreover assumes assumes that that

the the enzyme enzyme

is is

and and

one one

with with

= =

M M T50 T50 is is 5 5 10- X X

times as as times 3 effective. effective. The The concentration concentration 2 2 value value

is is

thus thus

5·6 5·6

times times 1O~ 1O~

X X

as as

efficient efficient

as as water, water, one one with with = = 1M 1M T50 T50 is is 55 55 times times as as effective, effective,

The The effective effective

concentration concentration

of of water water

is is 55·6M; 55·6M;

an an acceptor acceptor with with = = 10-3M 10-3M T50 T50

the the

value value of of

tj(l-t) tj(l-t)

is is ohviously ohviously

greatly greatly affected affected by by small small experimental experimental errors. errors.

than than

an an indication indication

of of the the

"true" "true" values. values. Apart Apart from from theoretical theoretical considerations, considerations,

polations polations

are are

therefore therefore

highly highly

uncertain uncertain

and and the the derived derived T T values values 50 50 are are no no more more

determined determined

at at

acceptor acceptor

concentrations concentrations

well well away away 50 from from T the the resultant resultant , ,

extra

Where Where

low low solubility solubility

or or inefficiency inefficiency

of of

an an acceptor acceptor has has meant meant that that t t must must

be be

as as

the the equations equations

in in Jermyn Jermyn

(1962a) (1962a)

suggest suggest that that they they will will at at the the ends ends of of the the range. range.

the the

linear linear

relationship relationship

holds holds

well; well;

beyond beyond these these points points deviations deviations become become

marked marked

this this

line line

cuts cuts

10g[t/(1-t)] 10g[t/(1-t)]

= =

O. O. Provided Provided that that < < 15% 15% < < t t 85%, 85%, approximately, approximately,

the the experimental experimental

points points and and

determined determined

T50 T50 as as the the concentration concentration value value at at which which

near near

enough enough

to to linear linear

for for

a a

number number

of of acceptors acceptors for for a a straight straight line line to to be be drawn drawn through through

would would

hold. hold.

As As Figure Figure

I I shows, shows,

the the

relationship relationship

between between log[t/(l-t)1 log[t/(l-t)1 and and log log c c is is

10g[t/(1-t)] 10g[t/(1-t)] = = Kl Kl log log c+ c+ K2 K2

takes takes

a a

generalized generalized

sigmoid sigmoid

form. form.

If If it it were were a a true true sigmoid sigmoid curve, curve, the the equation equation

observation observation

that that the the relation relation

between between

t t and and c c (the (the concentration concentration of of added added

acceptor) acceptor)

1962b); 1962b);

the the problem problem

of of

determining determining

empirically empirically T50 T50 may may be be simplified simplified by by

the the

acceptor acceptor

is is

complex complex

and and

involves involves

a a large large number number of of constants constants (Jermyn (Jermyn 1962a, 1962a,

The The expression expression

for for the the dependence dependence

of of t/(l-t) t/(l-t) on on the the concentration concentration of of added added

enzymically. enzymically.

No No significant significant

variation variation in in the the value value of of t t could could be be detected. detected.

concentration concentration

of of 5xlO-

M M

(0'05-50% (0'05-50%

of of the the total total glucoside glucoside 5 present), present), was was removed removed

mixtures mixtures

an an

approximately approximately

constant constant

amount amount

of of the the ,a-glucoside, ,a-glucoside, equivalent equivalent

to to a a

phenyl phenyl

,a-glucoside ,a-glucoside

at at initial initial

concentrations concentrations

over over the the range range lO- lO-~M. lO-~M. From From c c

these these

deliberate deliberate

experiment experiment

was was

set set up up

with with

the the acceptor acceptor methanol methanol (concn. (concn. 1M) 1M)

and and

the the

amount amount

of of phenyl phenyl

,a-glucoside ,a-glucoside

broken broken

down down in in any any experimental experimental series. series.

A A

are are constant constant

within within

experimental experimental

error, error, although although no no attempt attempt was was made made to to standardize standardize

with with

the the

usual usual

(2 (2 X X 10-3

M ) ) initial initial concentration concentration of of donor donor phenyl phenyl ,a-D-glucopyranoside, ,a-D-glucopyranoside,

t-butyl t-butyl

alcohol, alcohol,

hexane-I,6-diol), hexane-I,6-diol),

the the values values of of deduced deduced T50 T50 under under standard standard conditions, conditions,

Empirically, Empirically,

it it has has

been been found found

that that for for certain certain well-studied well-studied acceptors acceptors (ethanol, (ethanol,

controls controls the the kinetics. kinetics.

relative relative

affinity affinity

of of

PhOGlu PhOGlu

for for

the the

two two complexes complexes

[(k~ [(k~

+k8)/k_~ +k8)/k_~

as as against against n 7 (k +k

)/k_ 7

] ]

is is so so low low

that that

the the

enzyme enzyme

is is

essentially essentially

present present only only as as E. E. 2 H 0 0 or or E. E. ROH ROH and and the the

of of

[D]. [D].

Qualitatively, Qualitatively,

dependence dependence

of of on on [D] [D] T50 T50 will will occur occur only only when when [PhOGlu] [PhOGlu]

acceptor acceptor

concentration concentration

is is independent independent

of of

[D], [D], the the value value of of will will be be T50 T50 independent independent

value value

of of the the

v v constants constants

2 a

etc. etc.

In In

, , .•. .•. any any range range where where the the value value of of t/(l-t) t/(l-t) for for

any any

low low concentrations concentrations

of of donor, donor,

the the concentration concentration

range range involved involved depending depending

on on the the

with with

the the

net net conclusion conclusion

that that

t/(l-t) t/(l-t)

will will

become become visibly visibly dependent dependent on on [D] [D] only only at at

1 a +a [D]-1+a 2 [D]-2+ [D]-2+ ...... , ,

mathematical mathematical

analysis analysis

(Jermyn (Jermyn

I I

962a) 962a)

may may be be simplified simplified to to a a power power series series of of the the form form

The The

formula formula

that that

expresses expresses

t/(l-t) t/(l-t) in in

terms terms of of [D] [D] that that emerges emerges from from the the

:FUNGAL :FUNGAL CELLULASES. CELLULASES. XV XV 907 907 908 M. A. JERMYN

can be regarded only as a parameter of the reaction, giving a measure of acceptor efficiency, to which no physical reality is to be ascribed. The effect of added acceptor on overall enzyme kinetics may also be described in a qualitative fashion. Figure 2 illustrates the expected results in terms of Lineweaver-Burk plots. Although mathematical investigation shows that Lineweaver-Burk plots are not necessarily linear when both of two competing reactions are taking place to a significant degree, no departures from linearity have been observed in this study; certainly none such that the known degree of experimental error would justify any other procedure than drawing a straight line through the experimental points. The range of glucoside concentrations covered was probably not sufficient to produce noticeable effects.

------y . r 0 //7/;/.'~!-:::"~./ 7' ' -'"c=c o o "~ -1 · :~;71~--

-3 -2 -1 o LOG10 ACCEPTOR CONCENTRATION Fig. I.-Relation between t, the fraction of transfer, and acceptor concen tration for a variety of acceptors chosen to cover the range. Donor, phenyl ;3-D-glucopyranoside, initially at 4XI0-3M; pH 5·0 and 28°C. 1, decane- 1,10-diol; 2, pentan-l-ol; 3,2,2-dimethylpropan-l-ol; 4,2-methylbutan-2-ol; 5, methyl ;3-DL-arabinoside; 6, DL-butane-2,3-diol; 7, hexan-2-ol.

III. MATERIALS AND METHODS (a) General Considerations Reducing sugar was measured by the Somogyi-Nelson method (Nelson 1944), and phenol according to a modified version of the method of Folin and Ciocalteau (1927). For the survey experiment set out in Table 1, commercial samples of the acceptors were used as received, unless the blanks in either the sugar or phenol determinations were above the tolerable limit. In this case liquids were redistilled and solids recrystallized to give an acceptable product. Butan-2-ol was resolved according to Kantor and Hauser (1953), and pentan-2-o1 according to Pickard and Kenyon (1911). Commercial inactive butane-2,3-diol was used to prepare meso-butane-2,3-diol and DL-butane-2,3-diol according to Wilson and Lucas (1936), and commercial cyciohexane-1,4-diol to prepare the cis and trans isomers according to Perrine and White (1947). Many of the sugar derivatives were samples prepared previously in this laboratory for other purposes. All relevant details about the acceptors are noted in Table 1.

extraneous extraneous

polymers polymers

than than

non-induced non-induced

enzyme enzyme preparations preparations from from growing growing cultures. cultures.

of of

washed washed mycelium mycelium

(Jermyn (Jermyn

1965). 1965).

This This

material material is is in in any any case case much much freer freer of of

into into

which which

enzyme enzyme

had had been been secreted secreted

after after phenyl phenyl ,8-D- thioglucopyranoside thioglucopyranoside induction induction

without without

further further

purification, purification,

is is

a a dialysed dialysed

and and lyophilized lyophilized preparation preparation of of the the

medium medium

each each

occasion. occasion.

In In view view

of of

this this

result, result,

the the source source of of enzyme enzyme that that has has been been used, used,

each each new new

batch batch

of of enzyme enzyme

used used

in in

this this

study. study. The The same same finding finding has has been been made made

on on

that that

may may

be be

potential potential

acceptors acceptors

(Jermyn (Jermyn

1962c), 1962c), this this point point has has been been rechecked rechecked

for for

since since preparations preparations

that that have have

not not been been stringently stringently purified purified contain contain carbohydrates carbohydrates

This This

point point

has has been been

repeatedly repeatedly

checked checked

in in the the past past (cf. (cf. Jermyn Jermyn 1962b); 1962b); however, however,

trations trations of of very very

efficient efficient acceptors-"anti-competitive acceptors-"anti-competitive activation". activation".

m [H 2

0j. 0j.

h: h: ';> ';>

kl1 kl1 kg; kg;

the the

series series

of of parallel parallel

lines lines

are are typical typical of of the the situation situation

with with

low low

concen· concen·

with with steady. steady.

state state

favouring favouring E.GluOPh.H.O E.GluOPh.H.O

in in

e e and and E.GluOPh.ROH E.GluOPh.ROH

in in j. j. g: g:

Km[ROH] Km[ROH] > >

breakdown breakdown

of of which which

is is

not not

being being

measured. measured.

e,j,g,h: e,j,g,h:

s > > kl1 kl1 k . . e,1: e,1: Km[ROH] Km[ROH] < <

Km[H.O] Km[H.O]

diag diag

lostic lostic

for for

"anti.compe:itive" "anti.compe:itive"

inhibition, inhibition,

i.e. i.e. competitive competitive

inhibition inhibition

of of

that that substrate substrate

the the

E E

GluOPh, GluOPh,

ROH ROH

in in b. b.

c: c:

Km[ROH] Km[ROH]

m < <

2 K [H

0]. 0]. d: d: kll kll

0; 0; = = the the series series

of of

parallel parallel

lines lines

are are

kg> kg>

. . a,b: a,b:

Km[ROH] Km[ROH]

m > >

2 K [H 0] 0] with with

steady.state steady.state

favouring favouring E.GluOPh,H

0 0 in in a a and and

high high

concentrations concentrations

of of added added

acceptor; acceptor;

5 5

theoretical theoretical = =

reaction reaction with with

acceptor acceptor

only. only.

a,b,c,d: a,b,c,d:

acceptor acceptor

on on

Lineweaver-Burk Lineweaver-Burk

plots. plots.

Throughout, Throughout,

= =

1 1 no no added added acceptor; acceptor; 2,3,4 2,3,4 = =

low, low, medium, medium,

Fig. Fig.

2.-How 2.-How

various various

relations relations

between between

the the

kinetic kinetic

constants constants will will

alter alter the the effect effect of of added added

RECIPROCAL RECIPROCAL OF OF DONOR DONOR CONCENTRATION CONCENTRATION

5 5

0 0

I I ~ ~

(e) (e)

------nn------Gj------7;/(h) ---7;/(h) '" '"

u u

;:: ;::

0 0

z z

"' "'

> >

..J ..J

0 0

u u

t t

,. ,.

5, 5,

(a) (a)

a a

level level much much

above above

the the

working working

concentrations concentrations

(1-4 (1-4

1O- X X used used M) M) in in this this

study. study.

and and

glucose glucose from from aryl aryl

,8-D-glucopyranosides ,8-D-glucopyranosides

at at concentrations concentrations of of the the latter latter below below 0 0 '1M, '1M,

The The

aryl aryl ,8-glucosidase ,8-glucosidase

of of

S. S.

atra atra liberates liberates equimolecular equimolecular amounts amounts of of phenols phenols

phenyl phenyl

,8-D-glucopyranoside ,8-D-glucopyranoside

and and salicin salicin were were free free of of detectable detectable phenols. phenols.

detectable detectable reaction reaction

in in solution solution

up up to to concentrations concentrations

of of 1O- for for M M reducing reducing

sugar; sugar;

tory tory

preparations; preparations;

salicin salicin

was was

a a commercial commercial

(Fluka) (Fluka) product. product. All All of of them them gave gave no no

Phenyl.8-D-glucopyranoside Phenyl.8-D-glucopyranoside

and and p-nitrophenyl p-nitrophenyl

,8-D-glucopyranoside ,8-D-glucopyranoside were were labora

FUNGAL FUNGAL CELLULASES. CELLULASES. XV XV 909 909 o <0 ......

p: ?-- c..., ~ l':j ~ ><: Z

are

8-7

"thia-" 27

10 9·1 5·0 14 10

T50 12

34 12

58 73 210 12 33 100 290 350 410

170 240 23

380 220

260

480 300

3,500

and (10-3M

acceptors

1001

"oxa-"

glycol,

ethyl

Acyclic

glycol

galactitol

D-gillcitol

the

cellosolve

D-isomer length

L-isomer

28°0.

Oomments

Synonym

using

monoacetate

lactate

L-Arabitol

D-Arabitol

Xylitol Adonitol Diglycol Thiodiglycol and Ethylene Ethyl 99% 86%

D-Mannitol Dulcitol; Sorbitol;

chain

Oommercial

Butyl

Polyethylene

50) given

also

are

hexol

arrangement

',"·p=,.1

this

Name

dilution

-diol

,2,3,4,5,6-

to comments

1500

final

ACCEPTORS

-Pentan-2-01

-3-0xo-4-oxahexan-2-01

Thiapentane-l,5-diol

P'"'''ne·I,2,3,

[HO(OH.OH.O)nOH.OH.OH]

Hexane-l

DL-3-Methylbutan-2-01

3-0xapentane-I,5-diol 3- Pentane-I,5-diol

Pentan-I-ol 3-0xapentan-I-ol 4-0xo-3-0xapentan-I-ol

necessary DL-Pentan-2-01 D-Pentan-2-01 "L"

Pentan-3-01

Hexane-l,6-diol Hexan-l-ol DL-Hexan-2-01 "L"

Heptane-l,7 3-0xaheptan-I-ol Heptan-l-ol Octan-l-ol

Nonane-I,9-diol Decane-I,10-diol } Oal'bowax

}

assimilated

buffer

32 33 34 35 36 37 other 38 39 40 41 are No. 42 43

45 44 46

47 48 49 50 51

52 53 54

55 56 57 58 59 60

VARIETY

Compounds

any I)

TABLE

4·3

FOR

and

3,.4

42 55

79 7\0

90 74

Acyclic

240 580 170 26 100 65

320 150

480

140 IIO

470

1I0

300

170

1,100

ToO

4,000

1,000 heteroatoms

1,300

4,400

19,000

(IO-3M

citrate-phosphate 160,000

OF names

with

usual

alcohol

VALUES

5·4

alcohol

(McIlvaine

alcohol

More glycollate cellosolve

alcohol

acceptors

self-buffered

Oomments

D-isomer

5·0

Synonym

Glycerol

Pentaerythritol Tris,

Isobutyl Neopentyl

t-Butyl Erythritol

Pinacol

pH Methyl Methyl

98% Isoamyl t-Amyl

length;

nomenclature.

chain

Name

increasing

,B-D-glucopyranoside

order

1,3-diol

methylpropane-I,3-diol

phenyl

Methanol Ethane-I,2-diol

Propane-I,2,3-triol Ethanol Propane-I,3-diol 2,2-Dimethylpropane-l,3-diol 2,2-Di(hydroxYTIlethyl)propane

2-Hydroxymethyl-2-amino-

Propan-I-ol 2-Methylpropan-I-ol DL-Propane-I,2-diol 2,2-Dimethylpropan-I-ol

in Propan-2-01 2-Methylpropan-2-o1

meso-Butane-I,2,3,4-tetrol DL-Butane-l,2,4-triol meso-Butane-2,3-diol DL-Butane-2,3-diol 2,3-Dimethylbutane-2,3-diol

L-Butane-2,3-diol Butane-I,4-diol

Butan-I-ol DL-Butane-I,3-diol 2-0xo-3-oxabutan-I-ol 3-0xabutan-I-ol

DL-2-Methylbutan-I-ol

L-2-Methylbutan-I-ol 3-Methylbutan-I-ol

DL-Butan-2-01 I D-Butan-2-01 2-Methylbutan-2-01

1 2 3 4 5 7 6 8

~o.

10 II

12 13 14 16 15 17 18 19

21 20 Donor listed 22 23 25 24

26 27 28 29 30

31 No.

61 62 63 64

71 72

74 73

75 76 77

78 79

Benzyl Cyclopentanol Cyclohexanehexol trans-Cyclohexane-1,2-diol

Methyl

Methyl Potassium D

Potassium Potassium Potassium Potassium

Methyl Phenyl

-Arabono-

D-saccharate

alooho1

,B-DL-arabinopyranoside ,B-D-arabinopyranoside

,B-L-arabinopyranoside

a-D-glucopyranoside

,B-D-thioglucopyranoside

D-arabonate D-xy1onate L-arabonate

D-gluconate hydrogen

Name y-lactone

(one

isomer)

myolnositol

Equimolecular

Synonym mixture isorners

Comments

both

Sugars

(i0-3M

42,000

TABLE

1,300

Tso Cyclic

220

transfer

260

100

100 and 100 46 43

3 87 83

Compounds

(Continued)

No.

66 67 68

80 81 82

83 84

85 86

90 89

Compounds

Cyclohexane-l,4-diol

trans-Cyclohexane-1,4-diol cis-Cyclohexane-1,4-diol Cyclohexanol

Potassium Dipotassium D - p-Nitrophenyl Methyl

Methyl

Benzyl D-Glycero-D-gulo-heptonolactone Trehalose Potassium Melezitose

Galactono-

pyranoside

a-D-galactopyranoside

,B-D-fructopyranoside

D -

D-galactonate

lactobionate

Name

mannopyranoside

mucate y-Iactone

,B-D-thiogalacto

Commercial

isomers

Synonym

Comments

mixed

(10-

No No

T50

140 220 transfer

110 110 transfer transfer 830

transfer

120

360

130

q :z I-:rj

~ 0 t-<

trj t-< q

m t-< ~ trj rn ..q i><

'" ...... 912 M. A. JERMYN

The basic technique in transfer experiments was as follows. Phenyl {J-D glucopyranoside (10 mg) was dissolved in a solution (9 mI) containing water, acceptor, and buffer and equilibrated at the working temperature. At TO' temperature equilibrated enzyme solution (1 ml) was added and the reaction allowed to proceed for a predetermined time that depended on the enzyme preparation used and the effect, if any, of the acceptor on overall enzyme activity, but was calculated to allow about 10% decomposition of the phenyl {J-glucoside. At TO and Tx duplicate samples for each determination were pipetted into either the Folin-Ciocalteau or copper reagents, both of which immediately destroy enzyme activity; the rest of the procedure is described elsewhere (Jermyn 1962b). A group of experiments with different concentrations of the same acceptor was usually run simultaneously, together with a control without added acceptor, and a blank with the highest con centration of acceptor to make allowances for any reactive impurities present in the acceptor sample. Occasional small alterations in this procedure were necessary to achieve specific objects, but they were in all cases sufficiently obvious not to need to be set out in detail. The fraction of transfer is given by 1 moles of glucose liberated moles of phenol liberated ' since glucosyl residues transferred to form alkyl glucoside are not determined as reducing sugar, like those transferred to water. In practice, small variations in standards and reagents displace the control value of the ratio of liberated glucose to liberated phenol a little from unity in any given set of experiments. Provided this ratio was within the limits of 1·00 ±O· 03, the experiments were not rejected, and the observed control value of the ratio was used as a multiplier to correct the values of the ratio obtained in the presence of acceptor. p-Nitrophenyl {J-D-glucopyranoside, although it is the most convenient sub strate for measuring the concentration and general properties of enzyme solutions, cannot be used with any accuracy in experiments where glucose is to be determined by alkaline copper reagents. The glucose determinations become quite erratic, depending strongly on the heating schedule. The reduction of copper and of the aromatic nitro group are both possible reactions for glucose in hot alkaline solutions, and under the conditions of the determination they are apparently competitive. This substrate has therefore only been used in these studies for cases in which a small amount of potential acceptor was available. This was then tested under the normal conditions for estimating the Michaelis constant for p-nitrophenyl {J-glucosidase activity, and its pattern of reaction with the enzyme interpreted by means of Lineweaver-Burk plots (cf. Fig. 2). Similar considerations prevent the testing of reducing sugars as acceptors; they have been replaced in this study, not perhaps entirely homologously, by their simple glycosides. Nor can the most efficient acceptor discovered, pentaerythritol, be used in more precise kinetic studies because of its slow reaction with the Folin Ciocalteau reagent. This behaviour is not a consequence of impurities in the penta erythritol, but appears to be due to slow reversal of the normal method of synthesis of the molecule, under the conditions of the determination, to give formaldehyde.

w-diols, w-diols,

is is superimposed superimposed

a a much much

more more

specific specific

interaction interaction which which is is maximal maximal

at at a a

a a lower lower

value value

of of Tso Tso with with

increasing increasing

chain chain length length for for alkan-I-ols alkan-I-ols and and n-alkane-I, n-alkane-I,

It It

appears appears

from from the the

data data of of

Table Table 1 1 that, that, upon upon an an overall overall tendency tendency towards towards

I,IO-diol. I,IO-diol.

and and

Tso Tso of of

4·,5 4·,5

and and

12xIO-3M 12xIO-3M

for for

octan-I-ol octan-I-ol and and 5·7 5·7 and and ,5-3 ,5-3 IO-3 X X M M for for decane

for for nonan-I-ol nonan-I-ol

and and 1·5 1·5

1O- X X M M

for for undecane-I,ll-diol undecane-I,ll-diol 3 against against limiting limiting solubility solubility

solutions, solutions,

can can

be be extrapolated extrapolated

to to

give give approximate approximate

solubilities solubilities at at 20°0 20°0 of of 1· 1· 2 2

IO-3 X X M M

Henrikson Henrikson

(1952), (1952), used used

to to

calculate calculate the the molarity molarity of of octan-I-ol octan-I-ol and and decane-I,10-diol decane-I,10-diol

data data

of of Butler, Butler,

Thomson, Thomson,

and and Maclennan Maclennan

(1933) (1933) and and Ekwall, Ekwall, Daniellson, Daniellson, and and

The The limit limit to to

the the

substances substances

that that

can can be be investigated investigated is is set set by by solubility; solubility; the the

relationships relationships

can can be be covered covered in in a a finite finite series series of of tables. tables.

used used

in in

Table Table

1 1 is is

not not the the

only only

one one

that that could could have have been been used, used, and and not not all all

cross

glucopyranoside glucopyranoside

as as

the the donor donor

at at 28°0 28°0 and and pH pH 5 5 ·0. ·0. The The principle principle of of arrangement arrangement

Table Table

1 1 presents presents

a a comprehensive comprehensive

table table of of values values of of T50 T50 using using phenyl phenyl j3-D

(a) (a) T50 T50 Values Values

IV. IV. RESULTS RESULTS AND AND DISCUSSION DISCUSSION

Pentaerythritol Pentaerythritol

4·3 4·3 Undeterminable Undeterminable

Pentan-l,5-diol Pentan-l,5-diol

10 10 U U ndetermina ndetermina ble ble

3-Methylbutan-l-ol 3-Methylbutan-l-ol

65 65 4·2 4·2

Butan-l-ol Butan-l-ol

74 74 6·3 6·3

Propan-l-ol Propan-l-ol

320 320 63 63

Ethanol Ethanol

580 580 200 200

Methanol Methanol

llOO llOO 210 210

M M (IO-3 = = I) I) (IO-3 = = M M I) I)

Acceptor Acceptor

f3·D-Glucopyranoside f3·D-Glucopyranoside Salicin Salicin

for for T50 T50 Phenyl Phenyl Tno Tno for for

SELECTED SELECTED ACCEPTORS ACCEPTORS

J3.D·GLUCOPYHANOSIDE J3.D·GLUCOPYHANOSIDE

AND AND SALICIN, SALICIN, 28°0 28°0

AT AT pH pH AND AND 5· 5· 0, 0, AND AND CERTAIN CERTAIN

COMPARISON COMPARISON

OF OF

THE THE

VALUES VALUES

OF OF T50 T50 FOR FOR THE THE TWO TWO DONORS, DONORS, l'HENYL l'HENYL

2 2 TABLE TABLE

results. results.

absorption absorption

spectrum spectrum

from from the the

parent parent alcohols alcohols has has so so far far yielded yielded disappointing disappointing

photometry. photometry.

A A search search

for for alkyl alkyl

glucosides glucosides with with a a markedly markedly different different ultraviolet ultraviolet

in in which which

true true initial initial

rates rates can can be be

measured, measured, presumably presumably by by continuous continuous spectro

altering. altering.

Such Such systems systems

will will require require

another another type type of of experimental experimental approach approach altogether altogether

the the acceptor acceptor

is is

rapidly rapidly used used

up up

and and apparent apparent transfer transfer fractions fractions are are continually continually

acceptor acceptor

and and water water

takes takes

place place

only only

at at

acceptor acceptor concentrations concentrations

(1-5 (1-5 10- X X

M) M)

where where

in in

Table Table

2, 2, represent represent

in in fact fact

a a

Tso Tso so so

low low that that significant significant partial partial transfer transfer to to both both

where where

errors errors

are are

not not too too

great. great. For For salicin, salicin, the the values values marked marked "undeterminable" "undeterminable"

j3-glucoside j3-glucoside

the the

lowest lowest

values values

of of

found found T50 T50 4 4 (~ (~ 10- X X M) M) are are just just within within 3 the the

range range

if if this this is is

carried carried out out using using

acceptor acceptor

concentrations concentrations comparable comparable with with Tso. Tso. For For

phenyl phenyl

of of any any

acceptor acceptor

with with

a a low low enough enough

value value of of T T will will so so be be used used up up during during the the experiment experiment

A A more more

serious serious

limitation limitation

is is imposed imposed

by by the the fact fact that that an an appreciable appreciable fraction fraction

FUNGAL FUNGAL OELLULASES. OELLULASES. XV XV 913 913 914 M. A. JERMYN

chain length of 5-6 carbon atoms. The observed value of T50 may be the result of two interactions, a general one with the enzyme as a whole and a secondary one with a specific site. This observation may be joined by the further deductions that a primary hydroxyl group, a compact molecule that does not sterically hinder approach to this hydroxyl group, and the L-configuration favour a low value of T 50. The resulting prediction is that minimum values of T50 should be found for the L-isomers of such hexanols as 2,3-dimethylbutan-1-01, 2-methylpentan-1-ol, and 3-methylpentan-1-01. There is no evidence from the table that polyhydroxylic compounds, and specifically sugars, are necessarily more efficient acceptors. However, the variation in acceptor efficiency amongst compounds alike structurally but not sterically is so great as to suggest that steric factors may play an overriding part in the availa bility of these relatively rigid molecules as acceptors; the variation between methyl

> '" '"f- iii'" ~'" 2·8 >- f- ;; i= u '"w :!' ~ 2·4 Z w 2 "g

2-0'! ! ! ! 3·2 3·3 ! 3·4 3·5 3·6 RECIPROCAL OF ABSOLUTE TEMPERAT!JRE (X103 ) Fig. 3.-Enzymic breakdown of 1O-3M p-nitrophenyl ,B-n-glucopyranoside at pH 5· 0 in McIlvaine (citric acid-sodium phosphate) buffer at pH 5·0 in the presence of O· 5M pentaerythritol over the range 2-45°C. D- and L-arabinopyranosides or between methyl D-manno- and D-galactopyranosides is quite striking. If sugars are the "natural" acceptors such differences may be quite enough to regulate the type of syntheses performed by the enzyme without calling for high specificity for the acceptor. Table 2 illustrates the fact that the values of T50 for selected acceptors vary with the nature of the donor. The dependence of degree of transfer on the nature of the donor has already been adequately established (J€rmyn 1962b). Table 2 re-expresses these observations in terms of a new parameter. (b) Activation Energy of the Transfer Reaction Before any discussion of reaction mechanisms is attempted, it is necessary to be sure that a single mechanism is in fact operating over the temperature range studied. The most sensitive test would appear to be the construction of an Arrhenius plot; and Ebert and Stricker (1964) have shown breaks in the slope of the plot for the dextransucrase reaction corresponding to changes in mechanism. For a test

when when 1M 1M ethanol ethanol is is the the solvent. solvent.

pyranoside pyranoside

to to

ethanol ethanol

thus thus

requires requires

3000 3000 ~ ~ cal/mole cal/mole more more than than that that

to to

water, water,

to to

be be 2840 2840

cal/mole. cal/mole.

The The

transfer transfer

of of the the glucosyl glucosyl residue residue from from phenyl phenyl ,B-D-gluco

the the

slope slope

of of the the

line line

can can be be

used used

to to calculate calculate

a a

value value 2 for for 1 (E -E this this comes comes

); );

out out

the the reciprocal reciprocal

of of absolute absolute

tempera~ure, tempera~ure,

a a linear linear relationship relationship was was found. found. Moreover, Moreover,

set set

out out

in in Table Table

3; 3;

when when

the the logarithm logarithm

of of the the transfer transfer ratio ratio was was plotted plotted

against against

was was

measured measured at at

pH pH 5 5

in in 1M 1M

ethanol ethanol

over over a a temperature temperature range. range. The The raw raw data data

are are

An An experiment experiment

was was

set set up up in in which which the the transfer transfer to to ethanol ethanol from from phenylglucoside phenylglucoside

linear. linear.

the the

logarithm logarithm

of of

the the transfer transfer

ratio ratio and and

the the reciprocal reciprocal of of absolute absolute temperature temperature is is

acceptors acceptors

should should

depend depend

on on temperature, temperature,

in in such such a a way way that that the the relation relation between between

Hence Hence

if if all all other other

variables variables

are are fixed fixed

then then the the distribution distribution of of transfer transfer between between two two

10ge[tj(1-t)] 10ge[tj(1-t)] = = 10geZ+[(E2-E1)/R]· 10geZ+[(E2-E1)/R]· (l/T). (l/T).

then then

tj(l-t) tj(l-t) = = 2 1 Z.exp[(E -E )jRT], )jRT],

But But

this this

ratio ratio is is exactly exactly

the the transfer transfer ratio, ratio, t/(l-t), t/(l-t), discussed discussed earlier. earlier. If If

and and

R R is is

the the

gas gas

constant. constant.

The The ratio ratio

ofthe ofthe

rates rates then then has has the the 2 form form 1 Z.exp[(E

-E

)jRT]. )jRT].

2 Q.exp( Q.exp(

-E

/RT). /RT).

Here Here

P P and and

Q Q are are constants, constants, El El and and E2 E2 are are energies energies of of activation, activation,

with with its its

characteristic characteristic

rate, rate,

at at a a given given

temperature, temperature, 1 T of of P.exp( P.exp( , , -EljRT) -EljRT)

and and

acceptor, acceptor,

the the reactions reactions

can can

be be considered considered

quite quite generally generally as as two two processes processes each each

Whatever Whatever

view view

is is taken taken

of of the the

mechanism mechanism of of transfer transfer to to water water and and the the second second

be be

taken taken as as

typical typical for for the the enzyme enzyme and and all all substrate substrate pairs. pairs.

the the

conclusion conclusion

reached reached

may, may,

in in

the the absence absence of of strong strong evidence evidence to to the the contrary, contrary,

Since Since

the the

reaction reaction

used used was was

not not selected selected on on any any other other grounds grounds than than convenience convenience

involving involving

the the

formation formation or or decomposition decomposition of of ternary ternary complexes. complexes.

positive positive

choice choice

between between

the the reaction reaction

of of glycosyl glycosyl enzyme enzyme with with acceptor acceptor and and steps steps

E+GluOPhN0

2 2

EOGlu+N0 ~ ~

PhOH; PhOH;

but but the the data data will will not not allow allow us us to to

make make

the the

rate-determining rate-determining

step step

for for

transfer transfer

to to water. water. Therefore Therefore this this step step cannot cannot

be be

erythritol erythritol

exists exists

over over

the the

entire entire temperature temperature

range, range, and and that that it it is is different different from from

It It may may be be

concluded concluded

that that

a a single single

rate-determining rate-determining step step for for transfer transfer to to penta

transfer transfer

to to water water from from all all ,B.D-glucopyranosides ,B.D-glucopyranosides tested. tested.

the the

activation activation

energy energy

is is also also

outside outside

the the range range (7800-9300 (7800-9300 kcaljmole) kcaljmole)

found found

for for

energy energy

of of 7800±220 7800±220

kcaljmole kcaljmole

(Jermyn (Jermyn

1955) 1955) in in the the same same buffer buffer at at the the same same

pH; pH;

this this

glucoside glucoside

with with

water water as as

acceptor acceptor has has the the significantly significantly different different

activation activation

line; line;

the the activation activation

energy energy

is is 1l,250±100 1l,250±100

kcaljmole. kcaljmole. The The enzymic enzymic hydrolysis hydrolysis

of of

salicin. salicin.

The The

results results

are are presented presented

in in Figure Figure 3. 3. There There are are no no breaks breaks in in the the

Arrhenius Arrhenius

%) %) (below (below

1 1

for for phenyl, phenyl, p-nitrophenyl, p-nitrophenyl,

and and o-cresyl o-cresyl ,B-D-glucopyranosides, ,B-D-glucopyranosides,

and and

for for

free free reducing reducing

sugar sugar in in

the the

presence presence of of 0·5M 0·5M pentaerythritol pentaerythritol was was undetectable undetectable

would would

not not modify modify

the the

liquid liquid

environment environment

too too drastically. drastically. In In fact, fact, liberation liberation

of of

latter latter

should should

almost almost

totally totally

exclude exclude

water water from from the the reaction reaction at at concentrations concentrations

that that

side side was was

chosen chosen

with with

pentaerythritol pentaerythritol

as as acceptor, acceptor, since since Table Table 1 1 indicated indicated

that that the the

reaction, reaction,

the the

accurately accurately measurable measurable

decomposition decomposition of of p-nitrophenyl p-nitrophenyl p-D-glucopyrano

FUNGAL FUNGAL CELLULASES. CELLULASES. XV XV 915 915 916 M. A. JERMYN

The use of this difference to calculate a value for the energy of activation of the enzyme-phenyl glucoside-ethanol reaction that will be comparable to that for the enzyme-phenyl glucoside-water reaction depends on the assumption that 1M ethanol as solvent has had no general activating or depressing effect on the enzyme. Later papers in these series will demonstrate the amount of work needed to establish this point. The question can, however, be tackled empirically by observing the dependence of the calculated value of (E2 -E1 ) on ethanol concentration. At 0·3M ethanol the value of (E 2 -E1 ) came to 3140 cal/mole and at 0·1M to 3020 cal/mole, the same within experimental error as the value with 1M ethanol. There is thus reasonable justification for using the mean value of (E 2 -E1 ), 3000 cal/mole, and the known value (9300 cal/mole, Jermyn 1955a) for the water reaction, to calculate a value of 12,300 cal/mole for the enzyme-phenyl glucoside-ethanol reaction in a purely aqueous solution at pH 5.

TABLE 3 VARIAcrION iN THE TRANSFER BY THE j3-GLUCOSIDASE OF S. A'l'RA OF THE GLUCOSYL RESIDUE TO ETHANOL WITH VARYING Cl'EMPERATURE, USING 2 X 10-3M PHENYL j3-D-GLUCOPYRANOSIDE IN 1M ETHANOL AT pH 5·0 (MCILVAINE BUFFER)

Percentage Temperature Transfer (OC) Transfer to Ethanol ftatio ---_._------.. _--- --~--."-.------42 75 3·00 35 72 2·57 28 67 2·03 21 61 1·57 14 53 1·13 7 49 0·96 43 0·75

The rest of the studies set out in these papers have been carried out at the standard temperature of 28°C. It will be obvious that all the numerical values Ret out are no more than the values of temperature-dependent variables for this particular temperature. There is no evidflnce, however, that the nature of the effectR, aR opposed to their magnitude, is temperature-dependent.

(d) Choice of Acceptors for Further Study The considerations illustrated graphically in Figure 2 suggest the following acceptors as worth investigating, when they are combined with the empirical observations of the activating or depressing effects of various acceptors which were accumulated during the experiments leading to the T50 values of Table 1: (l) Activating, T 50 concentration low: i.e. the enzyme binds acceptor much more readily than water and the resulting complex is more active than that involving water. Hexane-1,6-diol was chosen for investigation since it was solid, highly soluble, and inert to the reagents.

WILSON, WILSON,

C. C.

E., E., and and

LUCAS, LUCAS,

H. H. J. J. (1936).-J. (1936).-J. Am. Am. Chem. Chem. Soc. Soc. 58: 58: 2396. 2396.

PICKARD, PICKARD,

W. W.

H., H., and and

KENYON, KENYON,

J. J. (19U).-J. (19U).-J. Chem. Chem. Soc. Soc. 99: 99: 45. 45.

PERRINE, PERRINE,

T. T.

D., D., and and

WHITE, WHITE,

W. W.

C. C. (1947).-J. (1947).-J. Am. Am. Chem. Chem. Soc. Soc. 69: 69: 1542. 1542.

NELSON, NELSON,

N. N. (1944).-J. (1944).-J. Bioi. Bioi. Chem. Chem. 153: 153: 375. 375.

Chem. Chem. 11: 11: 225. 225.

MIWA, MIWA,

T., T.,

TAKANO, TAKANO,

K., K., YASAMURA, YASAMURA,

A., A., SUZUKI, SUZUKI,

H., H., and and ISHIZAWA, ISHIZAWA, K. K. (1956).-Symp. (1956).-Symp.

Enzyme Enzyme

KANTOR, KANTOR,

S. S. W., W.,

and and

HAUSER, HAUSER,

C. C. R. R. (1953).-J. (1953).-J. Am. Am. Chem. Chem. Soc. Soc. 75: 75: 1744. 1744.

JERMYN, JERMYN,

lVI. lVI. A. A. (1965).-Aust. (1965).-Aust. J. J. Bioi. Bioi. Sci. Sci. 18: 18: 387. 387.

JERMYN, JERMYN,

M. M.

A. A. (1962c).-Aust. (1962c).-Aust. J. J. Bioi. Bioi. Sci. Sci. 15: 15: 769. 769.

JERMYN, JERMYN,

M. M.

A. A. (1962b).-Aust. (1962b).-Aust. J. J. Bioi. Bioi. Sci. Sci. 15: 15: 248. 248.

JERMYN, JERMYN,

M. M.

A. A. (1962a).-Aust. (1962a).-Aust. J. J. Bioi. Bioi. Sci. Sci. 15: 15: 233. 233.

JERMYN, JERMYN,

M. M. A. A. (1955b).-Aust. (1955b).-Aust. J. J. Bioi. Bioi. Sci, Sci, 8: 8: 577. 577.

JERMYN, JERMYN,

M. M. A. A. (1955a).-Aust. (1955a).-Aust. J. J. Biol. Biol. Sci. Sci. 8: 8: 563. 563.

FOLIN, FOLIN,

0., 0.,

and and CWCALTEAU, CWCALTEAU,

V. V. (1927).-J. (1927).-J. Bioi. Bioi. Chem. Chem. 73: 73: 627. 627.

. . EKWALL, EKWALL,

P., P.,

DANIELLSON, DANIELLSON,

1., 1., and and

HENRIKSON, HENRIKSON,

S. S. (1952).-Acta (1952).-Acta Chem. Chem. Scand. Scand.

6: 6: 1297. 1297.

EBERT, EBERT,

K. K. H., H.,

and and STRICKER, STRICKER,

H. H. (1964).-Z. (1964).-Z. Naturf. Naturf. 196: 196: 287 287 . .

CLELAND, CLELAND,

W. W.

(1963).-Biochim. (1963).-Biochim. Biophys. Biophys. Acta Acta 67: 67: 104. 104.

BUTLER, BUTLER,

J. J. A. A.

V., V.,

THOMSON, THOMSON,

D. D. W., W.,

and and

MACLENNAN, MACLENNAN,

\V. \V. H. H. (1933).-J. (1933).-J. Ch(,;ln. Ch(,;ln.

Soc. Soc. 1933: 1933:

674. 674.

BENDER, BENDER,

M. M.

L., L., and and

KEZDY, KEZDY,

F. F. J. J. (1964).-J. (1964).-J. Am. Am. Chem. Chem. Soc. Soc. 86: 86: 3704. 3704.

VI. VI. REFERENOES REFERENOES

The The author author

wishes wishes

to to

acknowledge acknowledge

the the technical technical assistance assistance of of Miss Miss Carol Carol May. May.

V. V. AOKNOWLEDGMENT AOKNOWLEDGMENT

the the class. class.

of of

the the

two two complexes. complexes.

t-Butyl t-Butyl

alcohol alcohol was was the the simplest simplest representative representative of of

is is of of the the same same order order

of of

magnitude magnitude

and and enzyme-acceptor enzyme-acceptor is is the the less less active active

(4) (4)

Repressing, Repressing,

concentration concentration

T50 T50

high: high: i.e. i.e. the the binding binding of of water water and and acceptor acceptor

value value amongst amongst simple simple compounds. compounds.

of of the the

two two

complexes. complexes.

Methanol Methanol

was was

chosen chosen as as having having the the highest highest

T50 T50

is is of of the the same same order order

of of

magnitude magnitude

and and enzyme-acceptor enzyme-acceptor is is the the more more active active

(3) (3)

Activating, Activating,

T T

concentration concentration

50 50

high: high: i.e. i.e. the the binding binding of of water water and and acceptor acceptor

ented ented by by benzyl benzyl p-fructopyranoside. p-fructopyranoside.

The The extreme extreme

case case where where

there there

was was no no transfer transfer to to the the repressor repressor was was repres

that that

involving involving

water. water.

Ethyl Ethyl

lactate lactate was was the the only only simple simple example example found. found.

more more

readily readily

than than

water water

and and

the the resulting resulting complex complex is is less less active active than than

(2) (2)

Repressing, Repressing,

concentration concentration

T50 T50 low: low:

i.e. i.e. the the enzyme enzyme binds binds acceptor acceptor much much

FUNGAL FUNGAL CELLULASES. CELLULASES. XV XV 917 917