γ-Glutamyl-cysteine synthetase from bovine lens

ARCHIVES

OF

BIOCHEMISTRY

AND

122, 73-84 (1967)

BIOPHYSICS

r-Glutamyl-Cysteine

Synthetase

II. Cysteine

Analogue

WILLIAM Department

of Ophthalmology,

Universiiy

Received

April

from

Bovine

Lens

Studies’

B. RATHBUN

of Minnesota

Medical

7, 1967; accepted

School, Minneapolis, May

Minnesota

55456

5, 1967

with L-gluSeventy-one compounds have been tested for their ability to NJUple tamic acid to form a r-glutamyl peptide bond through the action of a purified preparation of r-glutamyl-cysteine synthetase from bovine lens. The most active amino acids in the system were L-cysteine, L-c+aminobutyrate, and their esters, b-chloro-Lalanine, S-methyl-L-cysteine, L-cycloserine, L-norvaline, allothreonine, allylglycine, p-aminoisobutyrate, and L-homocysteine. The specificity pattern displayed by the enzyme allowed certain conclusions to be drawn concerning its requirements for the cysteine moiety. Several of the isolated enzymic product,s were hydrolyzed, and formed glutamic acid and the original cysteine analogue. The values for the apparent k’,,, and maximal velocity were determined for many of the reactions.

This preparation was used t,o study the activity of opOica1 isomers, derivatives, and analogues of glut,amate and cysteine in the system. The results indicated t’hat all I)isomers tested were inactive in the system, and those of cysteine and ar-aminobutyrate did not’ have an inhibitory effect,. Although the specificit,y for glutamate appeared absolute with regard to chain length, at least three methyl and hydroxy analogues of glutamate were acceptable in varying degrees to the enzyme. In recent years, a rather large number of peptides containing a y-glutamyl peptide bond have been isolated from bot’h plant and animal sources, including lens. Which of these compounds may arise through the action of r-GCS is unknown. The purpose of this study was t,o examine t)hc specificity of r-GCS for the cysteine moiety in detail.

The mammalian enzyme, T-GCS,~ which catalyzes the first step of glutathione synthesis, coupling L-glutamic acid and L-cysteine to form y-glutamyl-cysteine, has been purified from hog liver (l-3) and from bovine lens (4). Purified r-GCS from liver was used to replace L-cysteine with a small series of Lamino acids in the regular assay system of Strumeyer (2). analogues of gutathione were formed in lens extracts by substitution of a-aminobutyric acid, alanine, or threonine for cysteine, together with the other required components of the reactions (5). The study of the formation of y-glutamyl-cr-aminobutyrate in lens and liver extracts was complicated by its breakdown by y-glutamyl lactamase t,o form 2-pyrrolidone-5-carboxylic acid and cY-aminobutyric acid (6, 7). The lactamase was shown t’o be absent in the purified bovine lens r-GCS preparation (4). * This investigation was research grant NB-O1979-07, Service; and by a gift from Inc. used: 2 Abbreviations cysteine synthetase; Tris, aminomethane.

EXPERIMENTAL

supported in part by U. S. Public Health Alcon Laboratories,

PROCEDURE

S-(1,2-dichlorovinyl)-L-cysteine was prepared et following the general procedure of McKinney al. (8) ; S-(1,2-dicarboxyet.hyl)-L-cysteine by the procedure of Calam and Waley (9); and S-sulfoI>-cysteine by a modification of the procedure of Clark (10). All other amino acids were from com-

r-GCS, r-glutamyltris(hydroxymethyl)73

74

RATHBUN TABLE

ACTIVITY Amino acid

OF S-SUBSTITUTED

I

CYSTEINE

IN THE

SOWCC

-pGCS

SYSTEM Relative activityb

Structural fomlula

.R=HOOC-CH-CH,L-Cysteine S-Methyl-L-cysteine S-Ethyl-L-cysteine

A A A

NA* R-SH R-S-CH, R-S-CH&Hz 0

100 43 7.5

S-Carboxymethyl-L-cysteine L-Cysteine sulfinic acid L-Cysteic acid s-Sulfo-L-cysteine S-Carbamyl-L-cysteine< S-(1,2-dicarboxyethyl)-L-cysteine

E A A K E K

R-S-&H, R-SO,H R-SOaH R-S-SOaH R-S-O-CO-NH, R-S-CH-CHICOOH

0 1.6 0 0 100 0

S-(1,2-dichlorovinyl)+cysteine

K

A OOH R-S-CCI=CHCI

S-Benzyl-L-cysteine

B

R-S-CH,

S-Allyl-L-cysteined

E

0.6

/ -0 R-S-CHrCH=CHz

\

0 17

Q For this, and succeeding tables: A, Sigma Chemical Co.; B, K & K Laboratories, Inc.; C, General Biochemicals; D, Cycle Chemical Corp.; E, Nutritional Biochemicals Corp.; F, Mann Research Laboratories, Inc.; G, Calbiochem; H, Aldrich Chemical Co.; I, Distillation Products Industries; J, Mallinckrodt Chemical Works; K, Synthesized by author, see Methods section. b Activity expressed pmoles inorganic phosphate released from ATP in routine assay, in this and Tables II-IX. c Reported to be in rapid equilibrium with cysteine at this pH (11). d Difficult to obtain reproducible values; reaction rapidly slows down. This compound was not homogeneous in paper chromatography. mercial sources as noted in the tables. Amino acids reported in this study were homogeneous by paper chromatography unless noted otherwise. The following were recrystallized prior to use: L-arginine, L-histidine, and S-allylcysteine. Lens -/-GCS was prepared as described (4), and Fraction IV (purified 230- to 266-fold) was used throughout this study. The sources of other materials were the same as previously described (4). The general cysteine analogue survey was performed under conditions approximating apparent second-order kinetics with respect to ATP, analogues were 0.01 M with i.e., the cysteine respect to the L-component. The stock solutions of several amino acids having a tendency toward instability were made up and the pH was adjusted immediately prior to the enzyme assay. In no case, however, were solutions of amino acids made up earlier than 2 hours before they were assayed. Each cysteine analogue was assayed on no less than two occasions, each assay consisting of triplicate sample and blank tubes. The blank tubes contained the complete enzyme system except for glutamic acid. All other procedures used

herein were series (4).

described

in the first

paper

of this

RESULTS

Table I lists several S-substituted cysteine compounds and their relative ability to enter into the eneymic reaction. The results for X-carbamyl-L-cysteine were most probably a result of the rapid equilibrium which this compound exhibits with L-cysteine at this pH (11). Otherwise, the presence of oxygen attached or adjacent to the sulfur appeared to make the derivative inactive in the yGCS system. The value for S-allylcysteine is the average of several determinations. The reaction was poorly reproducible and the reaction velocity diminished rapidly with time. An attempt to prepare enough of y-glutamyl-S-allylcysteine to demonstrate by paper electrophoresis was unsuccessful (Table X) . Beta-substitution on t.he alanyl radical

?-GCS:

CYSTEINE

ANALOGUE TABLE

ACTIVITY

II

OF @-SUBSTITUTED ALANINE

Amino acid

SOUP+

73

STUDIES

COMPOUNDS IN -pGCS Structural fomlula

SYSTEM Relative activitf

R=HOOC --KCH*-1 n-Alanine L-Serine 0-Methylserineb 0-Phosphoserine L-Aspartic acid L-Leucine

A A B A A A

R-H R-OH R-0-CH, R-0-POJHZ R-COOH R-CH(CH&

7.1 0.4 1.4 0 0 1.5

n-Phenylalanine

A

0

DL-Allylglycine &Chloro-n-alanine L-a-Aminobutyric

D D A

R/j 0 R-CH=CHt R-Cl R-CH, HC-N

acid

L-Histidine n-Cysteine

F A

31 63 69 ‘CH

II R-C-NH R-SH

0 106

(1See footnote a, Table 1. b Not homogeneous in paper chromatography.

caused highly variable levels of acceptability to the enzyme (Table II). The isopropyl, phenyl, and imidazole groups were unsatisfactory. However, P-substitution of certain electron-attracting groups such as chlorine, vinyl, and methyl groups formed highly acceptable substrates. Oxygen in the pposition did not allow the analogue to act as a substrate. This observation has an exception in the threonine series (Table III). Gamma-substitution on a-aminobutyric acid (Table III) also produced variable results. An increase in chain length appears to have had a great effect upon the activity; addition of one methylene group to cysteine forming homocysteine, reduced the activity to about a third that of cysteine. Replacement of the terminal hydrogen of homocysteine with a methyl group to form methionine destroyed activity with this enzyme, in contrast, to S-methylcysteine (Table II). Addition of an amino or hydroxyl group in the omega position also destroyed activity. However, when the hydroxyl was not attached to the terminal carbon, as in threonine, the activity was much greater than Ohat of serine. More significant, however, was the fivefold increase in activity

which occurred when the hydroxyl group was erythro rather than threo, with respect to the amino group as in allothreonine. The straight-chain series of amino acids of Table IV indicate that a-amino-butyric acid, which forms part of ophthalmic acid, was the optimal length. Methyl substitution at the a-hydrogen position of ar-aminobutyrate, and other amino acids, inactivated the compounds (Table V). In only one case was an a-methyl amino acid found to be active: that was DL+ aminoisobutyric acid (Table VI). With this one exception, amino acids having the amino group in the p- or y-position were not active analogues in this system. Comparison of the data for t’he p-amino acids P-aminoisobutyrate, p-alanine, isoserine, and P-aminobutyrate demonstrates the a-methyl group was apparently required for activity. The lack of activity of isovaline (Table V) would indicate that the methyl and amino groups must not be on the same carbon for activity. Perhaps, then, a single a-hydrogen is a requirement. Methyl substitution on the P-carbon of cr-aminobutyric acid, threonine, or norvaline formed inactive analogues (Table VII). In-

76

RATHBURN TABLE ACTIVITY

III

OF -~-SUBSTITUTED a-AMINOBUTYRIC

Amino acid

ACID COMPOUNDS

sourcea

IN

y-GCS

Structural formula

Relative

R=HOOC-VH-(CHz),-

L-Threonine

acid

A A A

R-H R-CHz R-CHz-CH3

(allo-free)

C

HOOC-T-frCHa

activitya

L-Cysteine = 100

tiH* L-a-Aminobutyric L-Norvaline L-Norleucine

SYSTEM

69 27 0 5.4

NH, H nL-Allothreonine

(L-form shown)

B

L-Homoserine L-2’,4-Diaminobutyric acid L-Ornithine L-Lysine L-Homocysteine L-Methionine nL-Methionine sulfoxide L-Ethionine L-Arginine

F

HOOC-T-!-C&

26

tiH, bH R-OH R-NH, R-CHZNH? R-CHz--CH2NH2 R-SH R-S-CH3 R-SO-CHI R-S-CHz-CH3

1.4 0 0 0 29 0 0 0

R-CH 2-NH-!!NH

0

*

a See footnote a, Table I. TABLE ACTIVITY Amino acid

OF STRAIGHT-CHAIN S0UKC.P

IV

AMINO ACIDS IN y-GCS SYSTEM Structural formula

R=HOOCGlycine L-Alanine L-cu-Aminobutyric L-Norvaline L-Norleucine

A A A A A

acid

R-H R-CH3 R-CHz-CH, R-(CH,),-CHI R-(C&)3--CH,

$

ci NHz

Relative activitp

L-Cysteine = 100 0 7.1 69 27 0

a See footnote a, Table I. activation

of

Activity

occurred with of both @-hydrogens,

L-cysteine

methyl substitution as in penicillamine.

measurements

of analogues

con-

taining a p-hydroxyl group may be compared to those of L-cycloserine (Table VIII). In this compound, oxygen on the P-carbon is in a fixed steric position with respect to the amino group, and the enzyme exhibited

about the same activity with it as with allothreonine. L-Cycloserine was the only cyclic amino acid tested which was active with y-GCS; the n-isomer was not active (4). The inactivity of compounds with a secondary amino group was consistent with previous data of Tables V and VI, in which only compounds having a single a-hydrogen were accepted.

yGCS:

CYSTEINE

ANALOGUE

TABLE ACTIVITY

OF CYSTEINE

Amino acid

ANALOGUES salrd

WITH

77

STUDIES

V ~-METHYL

GROUP

IN

y-GCS

Structural formula (only L-form shown)

SYSTEM Relative

activity”

L-Cysteine = 100 CHz m,-a-Aminoisobutyric

acid

DL-Isovaline

A

HOOC- ’ -CH, 7 NH2 CH,

0.6

B

HOOC-A-CH2-CHa

0

ISH, & ”

B

DL-Lu-Methylserine

a-Methyl-nL-aspartic

acid

0

HooC-7-CH20H NH* YH3 HOOC--C-CHz-COOH

A

0

NH2 a See footnote a, Table I. TABLE CYSTEINE

ANALOGUES:

Amino acid

ACTIVITY

VI

OF p- AND

Sources

r-AMINO

ACIDS

Structural formula

Isoserine

E

@-Alanine DL-p-Aminoisobutyric

acid

A D

DL-p-Amino-n-butyric

acid

D

AH HOOC-CH,-CHzNH, HOOC- H-CHZNH, 7 CH, HOOC-CHz-CH-CH,

A

AH* HOOC-(CH&-NH,

r-Amino-n-butyric

acid

HOOC-CH-CHtNHz

IN

-pGCS

SYSTEM Relative activity0

L-Cysteine = 100 0 1.5 27 0 0

0 See footnote a, Table I.

Results from alteration of the carboxyl group are tabulated in Table IX. To avoid as much hlvdrolysis as possible, the amino acids containing a carbonyl group were dissolved just prior to their inclusion in the enzyme reaction mixture. The amides list,ed in this table, including serineamide, were all inactive. Cycloserine was the only active amide tested. That the carbonyl group itself might be required for activity was indicated by t,he lack of activity of 2-aminobutanol and 2-amino-butane, and of various amines which lacked t’he first carbon atom completely. Hydroxylamine, which has been

widely used to substitute for ammonia or an amino group for reaction with an active intermediate such as found in glutamine synthetase (la), or glutathione synthetase (13), was consistently near blank values wit’h r-GCS over a wide concentration range. Isolation of e?lxymically formed dipepticles. Preparative enzymic synthesis of several r-glutamyl dipeptides was performed under conditions approaching maximal velocity (4). The solutions contained L-glutamic acid, 0.015 M; cysteine analogue, 0.01 M (with respect to each isomer in racemic mixtures) ; ATP, 0.015 M; MgSO4, 0.02 M; and Tris-

78

RATHBUN TABLE CYSTEINE

ANALOWES:

VII

ACTIVITY OF BRANCHED-CHAIN

Amino acid

Source”

AMINO

ACIDS IN -&CS

Structural formula R=HOOC-CH-

SYSTEM

Relative activitya L-Cysteine

= 100

NH, .L-Valine

A G

DL-@-Hydroxyvaline

R-CH(CH,),

0 0

R-COH(CH,), H

L-Tsoleucine

A

R-C+H,ca

0

C& &Methyl-DL-aspartic

acid*

L-Penicillamine a See footnote a, Table I. * Not homogeneous in paper

A

R-CH(CH&COOH

0

G

R-CSH(CH,),

0

chromatography. TABLE

CYSTEINE

ANALOGUES:

VIII

ACTIVITY OF CYCLIC AMINO SourcP

Amino acid

ACIDS IN y-GCS

Structural formula

SYSTEM Relative activity” L-Cysteine

= 100

L-Proline

k Hydroxy-L-proline

CRL-2-Pyrrolidone-5kwboxylic

acid

H

HOOC-

Hz F c=o

&H

0

‘N’ H CHz-CHNHz L-Homocysteine

C

thiolactone

g_&,O

0

L-Cycloserineb

(1See footnote a, Table I. * Contains a small amount

of serine

sulfate buffer, 0.10 M, pH 7.6; final volume was 0.325 ml. In addition, the L-homocysteine reaction mixture was 0.01 M with respect to dithiothreitol. All solutions were saturated with toluene and were maintained

at 30’ for 24 hours. At this time, an aliquot was removed and diluted for orthophosphate analysis (Table X), and the remainder of the solution was deproteinized, separated by paper electrophoresis, and analyzed as previ-

&CS:

CYSTEINE

ANALOGUE

TABLE CYSTEINE ANALOGUES: ACTIVITY Analogue

STUDIES

IX

OF AMINES AND CARBOXYL DERIVATIVES Structural formula

SOUKP

CH&H*SHz HzNNHs NH,OH CH,CH-CH,

&Mercaptoethylamine

NH2 HS-CH,-CH*NH,

L-Cysteine methyl ester

HS-CH

-pGCS SYSTEM Relative

activity”

0

-!:C&H. 2A NG

F

IN

L-Cysteine = 100 0 2 2 0

Ethylamine Hydrasine Hydroxylamine Isopropylamine

L-Cysteine ethyl ester

79

on 3 0

HS-CHI-1‘-C(O-CH*-CHa

88

Ib L-Serineamide

A

L-a-Aminobutyric methylester

acid

D

L-a-Aminobutyric

acid amide

D

0

CH.1CH 2

55

0

nL-2-Aminobufanol

B

CH,CHzCH-CH,OH SHr

0

nL-2-Aminobutane

B

CHzCHzd H-CHa

0

a See footnote a, Table I.

ously described (4). The solution originally containing homocysteine was isolat,ed after previous reaction with an excess of Nethglmaleimide. Aliquots of the concentrated, isolated dipeptide were hydrolyzed wit’h 3 N HCI in a sealed tube at 103” for 4 hours. Examples of the bands obtained by paper electrophoresis are shown in Figs. 1 and 2 for the analysis of reaction mixt)ures containing allothreonine and p-chloroalanine. Table X shows the positions of each of the ninhydrin-positive zones resulting from such procedures. In all cases, with the exception of cycloserine, the isolat,ed dipeptide follow ing hydrolysis formed two ninhydrin-positive

bands which corresponded to glutamic acid and the original cysteine analogue. In the case of cycloserine, the hydrol>-sis product corresponded to serine. The cysteine analogues all had a net movement toward the negative electrode, which was quite rapid in t,he cases of P-amino-isobutyrate and cvcloserine. The enzymically formed dipept’lde usually migrated at a faster rate toward the posit’ive electrode than did glut,amate at pH 3.9. The exception was y-glutamylcycloserine. lIetermination

of values of apparent

I<,,,.

The cysteine analogues which were most active with the enzyme were maintained

80

RATHBUN TABLE PREPARATION

Cyst&e

AND

Yc Reactiona

analogue

L-a-Aminobutyrate DL-Allylglycine p-Chloro-L-alanine L-Cycloserine nL,-Allothreonine L-Norvaline nL-@-Aminoisobutyrate L-S-Methylcysteine L-Homocysteinec S-Allyl-L-cysteine

X

ELECTROPHORESIS

OF y-GLUTAMYL

Movement of zone (cm)b

Time of electrophoresis (hours)

Cysteine ~IKdOgW

16 8 5.5 3 6.5 4.8 15 9 16 4.5

-2.3 -0.8 -0.7 -8.8 -0.9 -3.6 -7.8 -1.0 -1.5 -0.5

85 80 68 60 58 53 53 52 33 1

DIPEPTIDES

dipeptide

glutamate

$6.0 f3.7 +2.3 +1.7 +3.5 f2.7 +5.s +4.1 +6.1 f2.8

f9.8 $6.4 +6.0 -0.6 +6.2 +4.0 $6.2 +8.4 +8.8 -

a Measured by ATP breakdown over the 24.hour period at 30”, pH 7.6. Calculated on basis of only one optical isomer being used. b Measured from origin to center of zone; (-) is movement toward the negative electrode at 4”. c Contained 0.01 M dit,hiothreitol. Before electrophoresis, was allowed to react with excess N-ethylmaleimide. under conditions that insured nearly maximal velocity and in which the initial reaction rate was first order with respect lo the enzyme (4). Velocit,y measurements were made at four t,ime intervals for each concentration of the analogues, and the initial velocity was determined by extrapolation to zero time. Lineweaver-Burk reciprocal plots (14) of each of these determinations are shown in Figs. 3-10. Table XI lists hhe K,,, and the maximal values for apparent velocity for each of these systems. IO I

5

Cm 0

5

DISCUSSION

A comparatively t,amyl peptides

IO 1

Allothreonine Enzyme Reaction Mixture Enzyme Product

Hydrolyzed Enzyme Product

FIG. 1. Paper electrophoresis at pH 3.9 of t.he enzyme reaction solution containing L-glutamate, nL-allothreonine, ATP, MgS04, and Tris-sulfate buffer. The enzymically formed band was eluted and concentrated; an aliquot was hydrolyzed as described in text.

from natu-

ral sources. Table XII shows that the formation of relatively few of these naturally occurring peptides can be explained as a result of catalysis by r-GCS enzymes possessing the specificity characteristics of the lens enzyme. However, these would include peptides having alanine, cr-aminobutyric acid, P-aminoisobutyric acid, S-methylcysteine, threonine, and perhaps S-allylcysteine in y-linkage to glutamate. Natural occurrence Cm IO

Glutamate

Isolated

large number of r-glu-

have been isolated

I

5 I

0 I

5 I

IO I

Glutamate @-Chloroalanine Enzyme Reaction Mixture Isolated Enzyme Product Hydrolyzed Enzyme Product

FIG. 2. Paper electrophoresis at pH 3.9 of the enzyme reaction solution containing L-glutamate, ,8-chloro-L-alanine, ATP, MgSO1, and Tris-sulfate buffer.

y-GCS:

CYSTEINE

ANALOGTJE

Sl

S’l?UI)IES

(44). Therefore, it is possible that t,he bulk of the peptides of Table XII are the result of glutamine synthetase act,ivitg, an enzyme which is lacking in the lens +33S preparation (4).

0

IO

30

20

IO?/3-Chloroolanine

0’

(Mole/Liter)-’

FIG. 3. Effect of p-chloro-L-alanine initial velocity of t,he r-GCS reaction

on at 37”.

the

0

2

4

6

8

IO

IOm?Cycloserine (Mole/Liter)-’ FIG. 5. Effect of L-cycloserine on the velocity of the y-(;CS reaction at, 37”.

-0

I

2

IO%-Methylcysteine

3

4

5

(Mole/Liter)-’

FIG. 4. Effect of S-methyl-L-cysteine on the initial velocity of the y-GCS reaction at 37”.

of X-sulfoglutathione and X- (1,2-dicarboxyet’hyl)-glutathione perhaps can be best explained as result’ing from the nonenzymic reaction of glutathione with thiosulfate or maleic acid, respectively. Perhaps most, or all, of t’he remaining peptides are formed as a result of y-glutamyl t#ransferase activity in which glutathione is utilized as a substrate and transfers the y-glutamyl bond to new acceptors. In view of the fat that highly purified enzyme preparat,ions of both plant and animal tissue sources cont’ain both transferase and glutamine synthetase activity, it is probable that. both activit’ies are due to the same enzyme

0

5

IO

IOm?Norvaline

15

20

10.4Allothreonine

25

(Mole/Liter)-’

FIN:. 6. Efl’ect of Id-norvaline on the velocity of the r-GCS reaction at, 37”.

2 J-LLLL! 0 5 IO

initial

15

20

initial

2’5

(Mole/Liter)-’

Fru. 7. EBect of DL-allothreonine on the initial velocity of the r-GCS reaction at 37”.

82

RATHBUN

10~3/Allylglycine (Mole/Liter)-’ FIG. 8. Effect of DL-allylglycine on the initial velocity of the enzyme reaction at 37”.

possible to arrange a position in which the p-amino group of n-/3-glutamine approximates a position attained by an a-amino group of L-glutamine. This has been used to explain the reactivity of p-aminoglutamic acid with glutamine synthetase, and the same argument might be used to explain the reactivity of /3-aminoisobutyric acid with y-GCS. The data demonstrate that oxygen on the p-carbon reduces activity with y-GCS to a very low level, unless this oxygen atom is in a defined steric relationship with respect to the substituents on the a-carbon, as in allothreonine and cycloserine. The most reactive substrates in the yGCS system (cysteine, fl-chloroalanine, and

r

I

I

I

2

10~3/pAminoisobutyrote

0

J 3

FIG. 9. Effect of DL-@-aminoisobutyric acid on the initial velocity of the enzyme reaction at 37”.

This study demonstrated that r-GCS is relatively nonspecific for the L-cysteine moiety. Electrophoretic isolation of eight dipeptides in this study and two in a previous study (4) confirmed the cysteine analogue survey based upon measurement of ATP cleavage. The specificity pattern was further investigated by determination of the values of the apparent K, for several cysteine analogues. These values were all higher than that of L-cysteine (IL = 0.44 X 104), presumably the natural substrate. The value for p-chloroalanine, however, was not much greater, both being readily used by the enzyme at relatively low concentrations. The acceptability of P-aminoisobutyric acid by the enzyme might be explained by the use of molecular models. Khedouri and Meister (4.5) have demonstrated that it is

IO

IO?Homocysteine

4

(Mole/Liter)-’

5

FIG.

velocity

(Mole/Liter)-’

10. Effect of L-homocysteine on t,he initial of the enzyme reaction at 37”. TABLE

VALUES

FOR APPARENT VELOCITY” Substrate

L-Cysteine* r,-a-Aminobutyrate” nL-Allylglycine P-Chloro-L-alanine L-Cycloserine DL-Allothreonine L-Norvaline nL-p-Aminoisobutyrate S-Methyl-L-cysteine L-Homocysteine

XI

K,

-END MAXIMAL

Apparent K,

(X

0.44 5.9 12 0.70 77 22 82 25 16 16

109

Maximal velocityC

57 44 33 66 41 29 55 30 42 21

a Obtained by the method of Lineweaver and (14) on the basis of only one optical isomer being used. * Values from (4). c Nanomoles/(liter) (min) (mg protein). Burk

?-GCS : CYSTEINE

ANALOGUE

TABLE NATURALLY

OCCURRING

83

STUDIES

XII I/-GLUTAMYL

PEPTIDES

Peptide

SOUVX!

r-GCS activitp

r-Glutamylalanine, -y-glutamylalanyl-glycine r-Glutamyl-fi-alanine r-Glutamyl-S-allylcysteine r-Glutamyl-a-aminobutyrylglycine r-Glutamyl-r-aminobutyric acid r-Glutamyl-b-aminoisobutyric acid r-Glutamyl-S-(p-carboxy-fl-methyl-ethyl)cysteineylglycine r-Glutamyl-S-(~-carboxy-n-propyl)-~.-cysteineylglycine -,-Glutamylethylamine r-Glutamylglutamic acid r-Glutamylglutamine r-Glutamylglycine r-Glutamylisoleucine r-Glutamylisopropylamine r-Glutamylleucine r-Glutamylmethionine r-Glutamyl-S-methylcysteine, S-methylglutathione r-Glutamyl-S-methylcysteine-sulfoxide r-Glutamylphenylalanine -,+Glutamyl-S-propylcysteine y-Glutamyl-S-(prop-l-enyl)-L-cysteine r-Glutamyl-8-(prop-1-enyl)-L-cysteinesulfoxide r-Glutamylserine y-Glutamylthreonineylglycine r-Glutamylvaline

Plants (15-17), brain (18)) lens (21) Crucifer (19), iris (43) Garlic (20, 26) Lens (5, 7, 21, 42) Crucifer (19, 22) Plants (19), brain (23) Onion (24)

7.1 1.5 17 69 0 27 -

Glutamine S-(1,2-DicarboxyethyI)glutathione S-Sulfoglutathione

Plants (15, 25) Tea leaves (27, 28) Brain (29) Brain (29) Brain (30) Onion (31, 32), urine (33) Crucifer (22) Plants (31, 34), urine (33) Onion (31) Plants (24,31,34,35), brain (18) Plants (34) Onion (24) Chives (36), garlic (37) Chives (38) Onion (39) Brain (18) Lens (5) Plants (15, 17, 31, 40), brain (18), urine (33) Ubiquitous Lens (9) Lens (41)

0 0 0 0 0 0 1.5 0 43 0 0.4 5.4 0 0 0 0

a Relative rate of formation of the indicated dipeptide by lens y-GCS, from data in this paper. L-Cysteine = 100. cr-aminobutyrate) were all alanine derivatives with electron-attracting groups of approximat!ely the same physical size, in the /!-position. A larger group of substrates which were less reactive also contained electron-attracting groups in the p- or yposition. The data are consistent with the requirement of a single or-hydrogen on the was substrate. A probable requirement demonstrated for a carbonyl group. A parallel may be drawn with the ,& eliminat’ion reactions catalyzed by metaldependent pyridoxal model enzyme systems. These systems react with many of the reactive substrates of the present series, including cysteine, 6 - chloroalanine, and

threonine (46). The mechanism of P-elimination required the ionization of the a-hydrogen, following coordination of the carboxyl group by a divalent metal ion. As the leaving group became more electronegative, the ease of cleavage increased in model systems. However, r-elimination was found to be quite sluggish. If the comparison with ,&elimination model systems is correct, then the carbonyl group, the a-hydrogen site, and the electronattacting group in the P-position would afford three possible sites of attachment of the substrate to the enzyme. This would offer an explanation for its stereospecificity while still allowing free access of the proposed

84

RATHBUN

y-glutamylphosphoanhydride (2, 3) to the amino group.

intermediate

ACKNOWLEDGMENT The author is indebted to Miss M. Virginia Betlach and Mrs. Caroline Golberg for assistance given during phases of this research, and to Dr. John E. Harris for his continued interest and support. REFERENCES 1. MANDELES, S., .~ND BLOCH, K., J. Biol. Chew 214, 639 (1955). 2. STRUMEYER, D. H., Ph.D. Thesis, Harvard University (1959). 3. STRUMEYER, D. H., A4~~ BLOCH, K., J. Biol. Chem. 236, PC27 (1960). 4. RATHBUN, W. B., Arch. Biochem. Biophys. 121, 000 (1967). 5. CLIFFE, E. E., AND WALEY, S. G., Biochem. J. 69, 649 (1958). 6. CLIFFE, E. E., .IND WALEY, S. G., Biochem. J. 79, 118 (1961). 7. CLIFFE, E.E., AND WALEY,~. G.,Biochem.J. 79, 669 (1961). 8. MCKINNEY, L. L., WE.\KLEY, F. B., AND ELDRIDGE, A. C., U.S. Patent 2, 890,246; Chem. Abstr. 63, 19917e (1959). 9. CALBM, D. H., AND WALEY, S. G., Biochem. J. 86, 226 (1963). 10. CLARKE, H. T., J. Biol. Chem. 97, 235 (1932). 11. STARK, G. R., J. Biol. Chem. 239, 1411 (1964). 12. KRISHN.IS\%-AMY, P. R., PBMILJINS, V., hND MEISTER, A., J. Biol. Chem. 237, 2932 (1962). 13. NISHIMURS, J. S., DODD, E. A., AND MEISTER, A., 1. BioZ. Chem. 239, 2553 (1964). 14. LINEWEAVER,~~.,AND BURK, D., J. Am.Chem. Sot. 66, 658 (1934). 15. VIRTANEN, A. I., AND MATIKKAL.~, E. J., Z. Physiol. Chem. 322, 8 (1960). 16. VIRTANEN, A. I., AND BERG, A.-M., Acta Chem. Stand. 8, 1089 (1954). 17. MORRIS, C. J., THOMPSON, J. F., AND ASEN, S., J. Biol. Chem. 239, 1833 (1964). 18. KA~azaw.4, A., KAKIMOTO, Y., NAKAJIMB, T., AND SANO, I., Biochim. Biophys. Acta 111, 90 (1965). 19. LARSON, P. O., Acta Chem. &and. 16, 1511 (1962). 20. VIRTANEN, A. I., AND MATTILA, I., Suomen Kemistilehti B34, 44 (1961). 21. WALEY, S. G., Biochem. J. 67, 172 (1957). 22. LARSON, P. O., Acta Chem. &and. 19, 1071 (1965).

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