Enzymatic mechanism of creatine amidinohydrolase as deduced from crystal structures

J. Mol. Biol. (1990) 214, 597-610

Enzymatic

Mechanism of Creatine Amidinohydrolase as Deduced from Crystal Structures Miquel Coll, Siegward H. Knof, Yoko Ohgat Albrecht Messerschmidt, Robert Huber Max-Plan&Institut fuer Biochemie 8033 Martinsried bei Muenchen, F.R.G.

Hans Moellering,

Lorenz Riissmann

Boehringer-Mannheim,

and Giinter Schumacher

Penzberg, F.R.G.

(Received 15 November 1989; accepted 23 March

1990)

Crystal structures of the enzyme creatine amidinohydrolase (creatinase, EC 3.5.3.3) with two different inhibitors, the reaction product sarcosine and the substrate creatine, bound have been analyzed by X-ray diffraction methods. With the inhibitor carbamoyl sarcosine, two different crystal forms at different pH values have been determined. ,4n enzymatic mechanism is proposed on the basis of the eight structures analyzed. The enzyme binds substrate and inhibitor in a distorted geometry where the urea resonance is broken. His232 is the genera1 base and acid, and acts as a proton shuttle. It withdraws a proton from water 377 and donates it to the NC,, atom of the guanidinium group. OH- 377 adds to the Cc,, atom of the guanidinium group to form a urea hydrate. Proton withdrawal by His232 leads to products. The reaction product sarcosine binds to the active site in a reverse orientation. The free enzyme was found to have a bicarbonate bound to the active site.

1. Introduction

ism of this reaction. Our approach was t’o analyze the three-dimensional structures of the enzyme complexed with substrate, product and different inhibitors. Creatinase has its pH optimum at 7.5.

The enzyme creatinase or creatine amidinohydrolase (EC 3.5.3.3) hydrolyses creatine to sarcosine and urea (Roche et al., 1950; Appleyard & Woods, 1956): NH II H,N-C-N-CH,-CO,H I CH,

+ H,O --) HN-CH,-C02H

0 II + H,N-C-NH,

AH, Creatine + water + sarcosine + urea.

It has been found in soil bacteria (Pseudomonas, Alcaligenes) and is involved in the degradation of creatinine by micro-organisms (Matsuda et al., 1986; Shimizu et al., 1986). Creatinine is first hydrolyzed to creatine by creatininase. The presence of creatinase has been reported in higher organisms and in human skeletal muscle but its metabolic role is unknown (Miyoshi et al., 1980a,b). The guanidinium group cleaved by creatinase is characterized by extreme hydrolytic stability. We are interested in discerning the enzymatic mechant On leave

from

The

National

Institute

of Health,

Department of Chemistry. Kamiosaka, Shinagawa-ku, Tokyo 141. Japan. 002%2836/90~140597-14

$03.00/O

597

The activity decreaseswith lower pH, being about 40% at pH 5.5 (Egloff, 1990). Crystals obtained at these two different pH values were analyzed. We previously reported the crystal structure of the monoclinic form of creatinase at pH 6 with the inhibitor carbamoyl sarcosine bound in the active site (HoelIken et al., 1988). There, two alternative enzymatic mechanisms were proposed. Here, we present the structures of the same enzyme and inhibitor at pH 7.4, in two crystal forms, the structure of the enzyme complexed with the substrate creatine at pH 7.4, with the inhibitor succinamic acid at pH 54 and pH 7.4, and with the product sarcosine at pH 5.4. We also report t,he structure of 0

1990

Bradcmic

Press

Limited

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M. Co11 et al

Table 1 Relative

inhibition

Inhibitor

constants

Formula

Zl,, bMY

NH Creatine

H,N-!-N(CH,)-CH,-COzH

7.0

Carbamoyl

0 II H,N-C-N(CH,)-CH,-COZH

0.22

H,N(CH&CH,-CO,H

1.2

sarcosine

Sarcosine

NH Acetamidinium chloride

H 21Y&H ’ ’

3 .HC’I

the decrease of the absorption of PTA at 250 nm following first-order kinetics. (‘reatinase was also found to cleavr the Se-C bond of the seleno analog of PTA. which was prepared by reacting chloroacetic acid with selenourea. Tn order to compare the ligands and inhibitors of the structural studies described below. we determined the inhibitor concentration [II,*] at half the initial rate of caleavage of PTA at 25°C in 0.1 M-phosphate buffer (pH 72). following absorption at 250 nm; the results are shown in Table I. Low solubility prevented the determination of A-,,, of PTA, so that’ these numbers provide a measure of the relative Ki values assuming competitive inhibition only. A value for the K, of creatine of 1.3 rnM (Yoshimoto et trl.. 1976) serves as a guide-line.

48

(b) Crystallization Succinic

HO-!,,, “2

acid

Succinamic

acid

H 21.-8,H

-CH 2~2 -(“O

‘2/Z -(‘H

H

0.50

-CO 2H

1.7

NH Dicyanodiamide 4.Aminobutyric S-(2.Aminoethyl)isothiuronium bromide

acid

H,N-J-NH-W

51

H,N-CH*-CH,-CHZ-CO,H

57

NH II H,N-C-S-CH,-CH,-NH,.

HBr

5.0

Bicarbonate a Bicarbonate shows deviations from a linear relationship the reciprocal initial velocity and inhibitor between concentration with stronger inhibition at high concentrations. The other inhibitors show linear behavior. For a definition of I,!,, see Materials and Experiments. section (a).

All crystals were obtained by bat,ch carystallization and micro-seeding. Crystallization drops were prepared b> mixing approx. 20 pl of protein solution (30 mg protein/ml in water) and 10 ~1 of huffer solution containing 20 26 (w/v) polyethylene glycol (PEG 6000). 0.2 M-sodium/potassium phosphate buffer (pH 5.4 or 7.4). 001 M-inhibibor, substrate or product. For the crystallization of the free enzyme only PEG and sodium potassium phosphate buffer was used. A slight precipitation was always obtained after mixing the protein solution with the buffer solution. The drops were stirred up to solution wit’h a glass needle previousI) soaked on a droplet t,hat. already cont’ained microcrystals. Large crystals grew in these drops after a frw days at 4°C. At pH 5.4. only a monoclinic form with space group p2, was obtained. having cell dimensions a = 60+X? ‘4. b = 11@55 A. c = 62.63 A (1 A = 0.1 nm) and b = 102W. At, pH 7.4. a trigonal form. space group P3,21, also appeared from t,he carbamoyl sarcosine inhibitor complex. The cell dimensions of the trigonal crystal wrre o=b=129.4a.c=624~andy=lZO”. (c) fntensity data collection

the free enzyme at pH 5.4 and a bicarbonate complex at pH 7.4. These experiments rule out one of the proposed enzymatic mechanisms, detail the other and confirm His232 as the acid and base involved in the hydrolysis of the guanidinium group of creatine.

2. Materials

and Experiments

Pseudomonas putida creatinase was cloned. expressed and purified as described by Hoefien et al. (1988). (a) Enzymatic

assays

The inhibition of creatinase by different substances was tested using a newly developed simple assay, based on the observation that creatinase cleaves the S-C bond of S-carboxymethyl-isothiourea or pseudothiohydantoic acid (PTA?). The products are urea and thioglycolic acid, respectively, which were detected by the appearance of blue color with ferric iron. Cleavage can be followed by

t Abbreviations used: PTA, pseudothiohydantoic acid; PEG, polyethylene glycol: CMS, carbamoyl sarcosine; r.m.s., root-mean-square.

All intensity data were collected on a FAST area detector-diffractometer from Enraf-Pu’onius (Delft. The Netherlands). The X-ray source was a Rigaku rotat,inganode generator operated at 5.4 kW. The apparent spot size was 0.3 mm x 0.3 mm. The focus-to-crystal distance was 350 mm. The data were evaluated on-line using thr MADKES system (Pflugrath & Messerschmidt 1987) applying specifications described by Huber et al. (I 987). The FAST int,ensity data were divided into batches of 65” crystal rotation and scaled to account for the change of t’he irradiated volume during the crystal rotation. This procedure also corrects for long-term detector instability and radiation damage. The data were corrected for absorption that was modeled by a 3-axis absorption ellipsoid (Huber & Kopfmann, 1969; Messerschmidt et al.. 1990). Table 2 shows the data collection statistics for the different crystals. (d) Patterson search of the trigonal

crystal ,form

A Patterson search (Hoppe, 1957; Rossmann & Now. 1962; Huber, 1965: Fehlhammer & Bode. 1975; Colman it al., 1975) was performed to solve the structure of the trigonal form using the structure of the monoclinic form as a starting model. The cell volume indicated 3 dimerir molecules in the cell, implying that the molecular dyad

Enzymatic

Mechanism of Creatinase

599

Table 2 Data collection statistics

Crystal 7CSAb CASA’ CREd SUCC e 7SUC f SACY g SARC h FREE i BIC7 j

Number of measured reflections 79,941 117,803 61.320 69,629 69,256 46,872 48,830 70,245 60,193

Number of independent reflections

R mergea of scaled and absorption corrected measurements

26,026 18,607 29,385 24,771 23,878 13,402 20,309 24,450 23,520

R mergea of Friedel pairs after averaging

6.5 7.0 92 8.5 7.7 65 50 11.5 8.4

3.9 31 63 $4 4.5 2.9 3.2 66 5.6

Completeness 99-2.99 89% 91.7 89.8 947 92.6 750 92.2 87.3 849

A

2.99-2.7

1A

(91,) 2.7 l-2.52

88.3 80.1 86.4 81.3 769 15.2 555 76.7 72.5

A

2.52-2.37

64.9 63.0 82.9 45.5 .io+i

41.5 32.2 58.7 254 26.0

31.2 58.5 4x.7

2.4 333 297

1\

w h ere I( is the observed intensity of reflection h in the ith measurement and (Z(h)) is the mean a Knew = ~l~(h)i-(~(h)>l~(~(h)); intensity of reflection h over all measurements of h. b Carbamoyl sarcosine complex (at pH 7.4), monoclinic; ’ carbamoyl sarcosine complex (at pH 7.4), trigonal; d creatine complex (at pH 7.4); e succinamir acid complex (at pH 54); f succinamic acid complex (at pH 7.4); g*h sarcosine complex (at pH 54): i free enzyme iat pH 54); j HCO; complex (it pH 7.4).

The model of was parallel to the x-axis and then 1 subunit was placed in a cubic cell of length 150 A. Triclinic structure factors were calculated from this model with resolution from 8 to 3.5 8, assigning a temperature factor of 20 A2 to all atoms. A model Patterson map was t’hen calculated with a 1 A grid spacing; 5000 highest peaks were selected from a radial shell of 3 to 20 A of the Patterson map. A crystal Patterson synthesis was also calculated using the 8 to 3.5 b data and a 1 A grid spacing. A cross-rotation function between the Patterson map of this model and the Patterson map of the crystal was computed using the PROTEIN computer package (Steigemann, 1974). Only an azimuthal search around the x-axis was necessary for the 2 possible solutions at 0, = 0” and 180”, respectively, and the rotation function was calculated for the Eulerian angles? & = 0” to 360” and e3 =O”, in 5’ steps. Two significant peaks resulted at 0, = 180”, f& = BO”, t$ = 0” and & = 180”, f& = 240”, 8, = 0”. The heights of the 2 peaks were 37.9 and 37.2, respectively, the next highest peak being 30.2 and the S.D. 0.3. A fine scan, with 1” steps. yielded 2 higher peaks at @I = 180”, & = 61”, Cl3= 0” and 8, = 180”, & = 241”, & = 0”. The monomer was oriented according to the results of the rotation function, and a translation function (Crowther & Blow, 1967) was calculated with programs written by Lattman (1985) and modified by J. Deisenhofer and R. Huber. For this, the Fourier transform of the oriented monomer was calculated, first using data from 8 to 3.0 A. Since there are 6 symmetry-related molecules in the unit cell, 5 translation searches were computed for pairs of symmetry related molecules 1-2, 1-3, l-4, l-5 and 1-6, where these numbers refer to the symmetry operations given by International Tables for Crystallography (1983). The maxima, 2nd highest peaks and the standard deviations of the 5 independent computations were. respectively: 4866, 211.7 and 38.9; 490.8, 206.6 and 38.9; 627.1, 538% and 56.3; 371.3, 265.6 and 48.9; 447.0, 210.2 and 33.1. All maxima were consistent and represent the cross vectors between symmetry-related coincides the dimer

with the z-axis in the trigonal was rotated so that the local

cell. 2-fold

t 0, rotates about Z, e2 about the new x-axis, OS about the new z-axis.

molecules. This allowed us to det’ermine the position of the molecule in the cell and to establish space group P3,21 as the correct enantiomorph.

(e) Re$nement Trigonal crystal form The oriented and positioned monomer model was refined as a rigid body with the Fourier transform fitting program TRAREF using 6 to 4 A data (Huber & Schneider, 1985), whereby the R-factor dropped from 47.2 y. to 38.1%. A combined crystallographic and energy refinement followed with the program EREF (Jack & Levitt, 1978) as implemented by Remington et al. (1982). The resolution was progressively increased in 3 steps: 8 to 3.5 A. 8 to 3 b and 8 to 2.5 A. After 15 c%yclesthe R value was 26.2%. At this stage maps with coefficients 2F0- FC and FO- FC were calculated, the model rebuilt and 43 water molecules added using a graphic terminal, Evans and Sutherland PS 390, and the program FRODO (Jones, 1978). A new round of refinement cycles was performed including a temperature factor refinement cycle before the model was again rebuilt and more water molecules were added. After 50 cycles of EREF, 5 rebuilding cycles and the addition of 184 water molecules. the R-factor was 16.0%. At this stage, the carbamoyl sarcosine was added in the active site and fitted in the residual electron density (see Fig. 2(c)). After 5 more cycles of EREF refinement. the final R-factor was 15.8 and the r.m.s. deviations from the ideal values were @017 A for the bond lengths and 2.5” for the bond angles. All Fourier maps were calculated using the PROTEIN program package (Steigemann. 1974). (i)

(ii) Monoclinic crystal forms All other structures were isomorphous with the original monoclinic crystal form. They were refined by EREF starting with the original model. Typically, about 8 cycles of EREF and 1 temperature refinement cycle were sufficient. However, in some cases a partial rebuilding of the molecule using Fourier maps was necessary, in particular for some side-chains. Note that there are small differences in the cell dimensions of the different crystals. Table 3 shows the results of the different refinements.

600

L?!.

et al.

Cdl

Table 3 Re$nement

(‘rystal

Resolution (4

T(‘S.4” (‘ASAb C’RE7’

8~0-25 8.0-2.6 X0-2.5

SI'CC'

d

8~0-23

7SlTC e

u.o-Pi

SA('Y

8~0-3~0

f

SARC’ g FREE h

8.0-2.5 8.0-25

lSTC'7 i

f+o- 2.4

Number of unique reflert,ions

Final (““l

R

Energ? (kcal/mol)

22 x09

18.3

- 8.528

04 IO

16:865 23,890 2" 05 1

I,58 17.4

18%

- 1403 -5.549

-5774

IWIT 0.0 l-4

I .!I 2-i ?:I

2;:659 12,377 19.260

Ii.5 16.4 17.X

- 6460 -577-l - 5890

0410 WOIO Of)1 2

I ,!I 2.2 2.2

dl.308 %1.,50-”

18.1 I ft.5

- 6399 --Cl91

04 IO 0.01 I

I3 23 1

0413

2.2

a Carbamoyl sarcosinr complex (at pH 7.4). monoclinic; ’ carbamo>-I sarcosine complex (at pH 7.4). trigonal; ’ c,rratine ~~nplrx (at pH 7.4): d succinamic arid complex (at, pH 5.4): ’ succinamic acid complex (at, pH 7.4): f,g sarcaosinr complex (a,t pH 5.4): ’ free Pnzvrnr (at pH 54): i HCO, complex (at pH 7.4). Note: I cnl = 4,184 ,J.

In the course of the refinement we noticed extra elecatron densit,y at residue Glyl86 in all t,he st,ructures. Interpreted as an arginine residue. this has been confirmed h? sequence analysis (unpublished results). (f)

Location

of the ,water

molecules

In the trigonal crystal, water moleculeswere incorporated using the following procedure. The asymmetric unit wit,h the enzyme monomer was filled with water molecules using the molecular graphics program MAIN written by I). Turk. All water molecules at) more than 3.5 A away from nitrogen or oxygen atoms were rejected. The remaining water structure was then energy minimized in the crystal environment’ with t,he program XPLOR (Briinger rt al.. 1987), keeping the protein atom positions fixed. For the optimization, all hydrogen atoms were placed at calculated positions. The inspection of the maps

revealed that many of the calculated water molecules were closet,o the electron density peaks.They wrerefitted in the density manually and incorporated gradually in the refinement. the water

After the energy minimization. about 40% of molecules were closer than 1 A to their final refined positions.This procedurewas found to be convenient and time-saving but cannot replace the visual

inspectionof the Fourier maps. 3. Results and Discussion Figure l(a) shows the substrate creatine with atom labels referred to in the text. The guanidinium group is planar due to the double bond character of the N-C bond, as shown in the crystal structure of creatine (Mendel & Hodgkin, 1954; Jensen, 1955). Figure l(b), (c) and (d) shows, respectively, the chemical formulas of carbamoyl sarcosine. succinamic acid and sarcosine. All three substances are inhibitors of creatinase and were co-crystallized with the enzyme as well as the substrate creatine. Sarcosine is a reaction product. Table 1 shows a comparison of the inhibition constants of these and other substances measured against the creatinase acid (PTA) as substrate pseudothiohydantoic defined before. Figure 2(a) to (j) shows the difference Fourier maps calculated with coefficients (Eb-fcl,) around

the active-site region of the different complex strucitures. These maps. except that of Figure 2(g), were calculated before incorporat,ing the ligands to avoid bias. The map shown in Figure Z(g) is an omit map calculated after removing the sarcosine molecule and refining a few cycles. W377 was also not included for the map calculation. It)s position is shown for comparative purposes. Figure 2(a) shows the structure with the inhibitor caarbamovl sarcosine (CMS: at pH 6). as described in our prevrous paper (Hoeffken et nl., 1988). The full3 refined CMS molecule in t,his enzyme -inhibit’or complex is shown fihted in the elect’ron density. An extra lobe of electron density emerges from the (‘(I) atom. This was interpreted as water (W377) reprcasenting the nucleophile at an intermediate st’abeof addition at, a distance of about 2.0 8. The c>arhamoyl moiety is non-planar and charact,rrized hy a dihedral C(I)-Nc3I angle of 80” and a tetra,hedrally distorted ?it3). In 19X8, we were uncertain about thca atom assignment within the c>arbamoylLwater complex. Clear evidence is now provided by analysis of the monoclinic form of the same complex at pH 74 (Fig. 2(b)). The density of W377 is absent. while the CMS molecule has maintained t)he nonplanar conformation. This experiment, rules out mechanism B of Hoeffken et CLZ. (1988): and cbonfirms scheme A. in which His232 is mainly responsible for all steps in catalysis act)ing as base a,nd arid. while

(0)

(b)

(cl

!di

Figure 1. C’hemical formulas of different molecules that bind in the active site of creatinase and whose caomplexrs have been analyzed: (a) substrate creatinr; (b) inhibitor carbamoyl sarcosine: (r) inhibitor sucncinamic acid: (d) product sarcosinr

Enzymatic

Mechanism

(b)

(d)

Fig. 2.

of Creatinase

601

602

M. Co11 et al

(el

.

’

h

h

(h)

Fig. 2.

L

Enzymatic

Mechanism

603

of Creatinase

lYi&L&. k

/ArgB335

Figure 2. Stereo difference Fourier maps of the active site of creatinase. All maps. except (a), which is an omit map, were calculated before adding any molecule in the active site. W377 was not included for the calculation. Electron density contours are plotted at 2.50 level. (a) CMS complex at pH 6. The refined CMS molecule is shown. An extra lobe of density in the carbamoyl end was assigned to a nucleophilic attacking water molecule, W377. (b) CMS complex at pH 74. monoclinic form (7CSA). The refined CMS molecule is shown in the density. Note that there is no density for W377. (c) Same as (b) but in the trigonal form (CASA). (d) Creatine complex at pH 7.4 (CRE7). The refined creatine molecule is shown in the density. (e) Succinamic acid complex at pH 5.4 (SUCC). The position of the CMS molecule and W377 as they are in the complex at pH 6, are shown. (f) Same as (e) but at pH 7.4 (7SUC). (g) Sarcosine complex at pH 5.4 (SACY). The refined sarcosine molecule is shown in the density. It occupies a reversed orientration. (h) and (i) Two views of thefree enzyme structure at pH 54 (FREE). The position of the CMS molecule and W377 as they are in the CMS complex at pH 6 (see (a)) are shown as thin lines. A bicarbonate molecule is shown as thicker lines occupying the triangular-shaped density. The other lobe of density was assigned to a water molecule W408. (j) Bicarbonate complex at pH 7.4 (BTC7). A refined bicarbonate molecule and a wat)er molecule, W405, are shown fitted in the electron density.

Glu262 and Glu358 serve an orientation function. Figure 3 is a schema of the active site in the CMS complex at pH 6 with all H-bonds and contacts less than 3.5 A. Figure 2(c) shows the structure with the CMS inhibitor at pH 7.4 in the trigonal form. It is very similar to Figure 2(b): and confirms the above. Figure 4 is a schema of the active site in this structure with all H-bonds. Figure 2(d) shows the electron density corresponding to t,he creatine complex at pH 7.4. The densit)y suggests a distorted guanidinium group similar to CMS. As in CMS at pH 7.4, there is no w377. Figure Z(e) shows the structure with the succinamic acid inhibitor at pH 5.4. For comparative purposes, the CMS and W377 molecules have been plotted, as in the CM&complex structure. It can be seen that the density of the succinamic acid covers almost all of the CMS molecule, with the exception

of the methyl group Cc,,. No density for W377 is present. Figure 2(f) shows the structure with succinamic acid at pH 7.4 and it is similar t’o that shown by Figure 2(d). Figure 2(g) shows the active site in the creatinase-sarcosine complex at pH 54. The density tits best to a sarcosine molecule in reverse orientation, in comparison with creatine or CMS (Figs 2(d) and 1). Both active sites, A and B, of the two sarcosine complex structures that we have analyzed (SACY and SARC in Tables 2 and 3) show similar electron density shapes. The carboxylate group facing ArgA64 and ArgB335 in the creatine and CMS complexes is now facing GluH262. However, it does not occupy the same position as the guanidinium group of creatine. Sarcosine is shorter and does not have the bulky amidino group, so that it may be displaced from GluB262 (see Fig. 6). Only one short distance O(*,(Sar - - - O”‘(GluH262) of 2.8 A (see Fig. 6) is found. GluB358 makes a hydrogen

604

M. Co11 et al. TyrB258 /

GluB262

3.5 __,...../.”

w,66..,..’

I

3,3

‘.._ 2.7 “..,.__

His 8232

w75

: !

::,,

2.7:. /

03

27

‘,,. ::

:

3,3

-_-_,--.

~,) .y -.....?,6 .-.....

GluB358

0 GluB358 s-

2.9

,/’

N\

E=N

,:’ ,,.’

2.6

-9~ .._,__,2.7 / PheA62 .-..._. .\, _,:’ ‘,\ ‘.,,3.1 .. .. ‘\, ,;-,’ w292 3.1 ,,;J..,,(3.3 “L N

I w292

I

kg8335

ArgA64

I

krg 8335

ArgA64

Figure 3. A schema for t,he active site U in the CM8 complex at pH 6. Hydrogen bonds are shown with broken lines, and a van der Waals contact between (Tc2,and PheA62 is shown. This diagram corresponds to Fig. 2(a).

bond with the Xc3) of sarcosine. This is made possible by a small displacement of the side-chain of this residue as indicated by an arrow in Figure 4. NE2of HisB232 is also H-bonded to the Oc2) of sarcosine. ArgA64 has rotated away from the posibion occupied in the creatine or the carbamoyl complex struct*ures to form a strong H-bond with AspAlOl. The binding of sarcosine in reverse orienbation at pH 54 indicates t’hat the guanidinium site (the inner site) is able to accept negatively charged substructures or t,o increase the normal pK of the carboxylate of sarcosine by about two unit’s, Figure 2(h) and (i) shows different views of the active

site

in the free

enzyme

at pH 54.

It

was

crystallized without added ligand. We found two lobes of density in the Fourier map in the active-site region. The density close to ArgA64 and ArgB335 could be assigned to a water molecule. The other lobe has a triangular shape. To analyze it further. we put a water molecule in the center of this densit) and, after some refinement. the difference Fourier map clearly showed extra density in the three corners of the triangle, suggesting a bicarbonate ligand. The binding of sarcosine had demonstrated that the inner site accepts carboxylate groups. Figure 2(h) and (i) shows the bicarbonate molecule fitted in the electron density. The position of the CMS molecule in the pH 6 complex is superimposed (thin lines). It can be seen that the bicarbonate superimposes on the carbamoyl group of CMS. However, it is rotated and displaced so that. the O(2)

Figure 4. A schema for the actiw sitca in the (‘MS complex at pH 7.4. trigonal form ((JA88). Hydrogen bonds are shown wit,h broken lines. This diagram corresponds to Fig. Z(c).

atom is closer to Hi&232 t,o make a hydrogen bond (see Fig. 7). The equivalent N,,, atom of creatine (Fig. 5) or the Oc3)atom of CMS are within H-bond distance to the 0” atom of GluB262 inst’ead. Figure 2(j) shows the active site of the HCO, complex at pH 7.4. The crystals were obtained 1)~ adding 75 mM-bicarbonate to the c:rFst,allization solution. Comparing this struct.ure with t,he f’reo enzyme at pH 5.4 (which also has a bicarbonatca group bound in the active site) it can be seen that the bicarbonate is displaced towards Arg64 and Arg335, making strong H-bonds with them and occupying the carboxylate or outs part oft he active> site (Fig. 8). W405 now occupies the guanidinium or inner site close to Glu262 and (9~358. Bicarbonate prefers the inner site c>loseto His232. Glud62 and CIu358 at low pH. and the outer site close to At-g64 and Arg335 at high pH, probably reflecting the titration of one of the groups involved, probably HCO; or His232. whose st)andard ph’ are closest to 6.

Figure 5 is a schema of the activcx sit,t. cvit)h creatine bound to it,. corresponding to the structurr shown in Figure 2(d). All H-bonds and caontacts of 3.5 A or less are shown. The tnolecule is tirmly held in basically t,hree points. The guanidinium group binds to GluB262 and GluK358: the carboxylatt~ group to ArgA64 and ArgB335. There is a van der Waals contact’ between the methyl group (:(2J and PheA62. Finally. NC2of Hi&232 is close to all three

Enzymatic

Mechanism

of Creatinase

.

605

His B376 Tyr B258 /

2-7 *‘.--.... HisB232

,a’

ASpA .. .. $...

W280

N , Y =NH2 / N

- , *, .* ,,a *.< NTii-

JNH2 \ N\

ArgB335 Arg A64 Figure 5. A schema for the active site, B, in the creatine with broken lines, and a van der Waals contact between Fig. 2(d). substructures of creatine. These interactions and the conformation of creatine and CMS bound to the enzyme are the basis of the enzymatic mechanism as suggest’ed below. Creatine and CMS bind to the enzyme in a conformation where the electron delocalization and resonance of the guanidinium and carbamoyl groups, respectively, are completely disrupted, since the planes described by

are almost perpendicular. The very high resolution analysis of the CMS at I.9 A documents this clearly, but also Figure Z(b), (c) and (d) indicate this structure unambiguously. Substrate and inhibitor adopt high-energy conformations using part of the binding energy to the enzyme. An attempt to fit creatine in the conformation found in single crystals into the

complex at pH 7.4 (CRE7). All hydrogen bonds are shown C,,, and PheA62 is shown. This diagram corresponds to

enzyme demonstrates this nicely and is shown in Figure 9. Atoms No,, ($2, a C(4) 1 O(,, and O(,, superimpose reasonably, but the guanidinium group deviates strongly. Bad contacts would occur with His232 and N(r) would be far from GluB262 and GluB358, whereas in the complex it is H-bonded to them. In the complex, N(,, is pyramidal with its lone electron pair pointing towards His NE2, as shown in Figure 2(a). The non-planar configuration of the guanidinium group leads to its polarization and increased and reduced positive charges at Cc,, and No,, respectively. This geometry resembles the expected transition state of the enzymatic reaction in which addition of a nucleophile to Ccl, is a central step. Indeed, such a structure is observed in the CMS complex at pH 6, where a water molecule or hydroxyl ion strongly interacts with the carbamoyl group. A tetrahedral adduct is not formed at neutral pH, however. This latter observation was the key to the identification of the nucleophilic water molecule, a problem left open in the first analysis (Hoeffken et al., 1988). It immediately rules out mechanism B of Hoeffken et al. and confirms

606

M . Cdl

et al.

TyrB258

GluB262 I

His B232 \

(

I B320

-L

GluB358

I

k\ Arg B335 Arg‘A64 Figure 6. A schema for the active site, B, in the sarcosine-creatinase complex shown with broken lines. The arrows indicate the displacement of some residues CMS complex structures. This diagram corresponds to Fig. 2(g).

at pH 5.4 (RACY). Hydrogen bonds arc in comparison with the creatine or the

GluB262 GluA262 I W327 2.6 ‘i

GluB358

2.7

HisA

-3.0

,: 2.7

GluA358

: :j

W408 ,’

2.9 ; N\

=N

d

:

3-l

‘.

‘,, 3.5

2.9,

“i-/N ‘\ N\

ArgA64

Figure

ArgB335

7. A schema for the active site B, in the free enzyme at pH 5.4 (FREE). Hydrogen bonds are shown as broken lines. A bicarbonate molecule and a water molecule, W405, occupy the position of the substrate molecule creatine. This diagram corresponds to Fig. Z(h) and (i).

W42

931.

w292

N\ i=, I ArgB64

,’

./’ : ‘.,, : 3 o ,‘.,,‘2-7 : ‘,.3.2 ..’ ,, ‘.(, 13.3’

‘N \

ArgA335

Figure 8. A schema for the active site. A. in the bicarbonate-creatinase complex at pH 7.4 (BIC7). Hydrogen bonds are shown as broken lines. This diagram corresponds to Fig. 2(j).

Enzymatic

Mechanism

607

of Creatinase

Figure 9. Stereo plot of active site, l3, of the creatine complex at pH 7.4 (CRE7). The distorted creatine molecule as it is in the complex is shown in thick continuous lines. The thin lines indicate the superimposed creatine molecule as observed in single crystals. The carboxylate group has been fitted, but the guanidinium end deviates st,rongly.

‘O,C-Glu262 .. ‘O,C-Glu262

H His232 ‘o,c-Glu358 “”

-O,C-Glu358

/ co;

(a)

(b)

‘O,C-Glu262 -O,C-Glu262

His232

‘. ‘o,c-Glu358 ‘..

-0,c-Glu358

Cc)

(d)

\ ___ ‘O,C-Glu262 His232

,W NH

Figure 10. Proposed enzymatic mechanism based on the 8 structures analyzed: (a) 1st proton transfer from a water molecule to His232; (b) breakage of the resonance of the guanidinium group and nucleophilic attack on C(r) by an OHion; (c) tetrahedral adduct; (d) protonation of N,,, and 2nd proton transfer to His232; (e) reaction products

AI. Co11 et al. mechanism A in which. as a principal feature, His232 acts as general base and acid, and proton shuttle. All complexes with creatinr and (‘MS show a hydrogen-bonding interaction between N” of His232 and SC,,, independent of pH and favored by the pyramidal distortion of NC3) projecting its lone elrcatron pair towards N”. This hydrogen bond and the disruption of the u~en TCSO~,LL~CP favors formation of the tetrahedral adduct by addition of W377 to C:,,, of CMS as its formation rrquires proton abstraction. probably by His232 (see Fig. IO of HoeEken et nl.. 1988). The tetrahedral adducat is a, stable intermediate only under acidic conditions and not seen at neutral pH. In the absence of information about the pK values of t,he polar groups in the active site, we do not offer an explanation for this but regard it as an indication that the equilibrium constant) for tetrahedral adduct forma,tion ma,v not be far from unity. The observation that the inner substrat’r binding site close to Glu262 and Glu358 a.ccommodat’es substructures of similar geometry but different polarities. the guanidinium (+) and carbamoyl (n) groups, documents very, clearly t)he potential to balance and distribut’e Internal charges over the network of many polar groups in hydrogen-bonding contact. This was oontirmed by t’he binding of thr carboxvlat,r (sarcosine) and bicarbonate groups. however. we emphasize our ignorance of the charges in t.hese cases. A reaction mechanism based on these considerat,ions is shown in Figure 10. (1) W377 and substrate are close to. or in, the binding pocket to form a pre-complex. His232 is unprotonated. (2) The substrate binds in distort’ed geometry. His232 is protonated by W375. (3) Hydroxyl 377 adds t’o (I,,,. (4) The substrate is protonated at XC3) 1,~ His232. (5) Proton abstraction from the guanidinmm hydrate to His232 leads to t’he collapse of thr tetrahedral structure and to products. The observed complexes of (areatine and CM9 at neut,ral pH may represent stage (2) while CMS at pH 6 reaches stage (3). In Hoeffkrn et al. (198X). mechanism A included inversion at nitrogen NC3). (loncomitant with the nucleophilic addition of OH- to a planar guanidinium group, the lone electron pair on ?i,,, develops anti-periplanar and must invert to establish a hydrogen bond with N” of His232. Tn the observed distorted geometry of substrate described here, tht lone elrc*tron pair is already directed towards His232 and no inversion is required. It appears that the transition st’ate of t,he react’ion changes with the pH. and occurs at a later stage at low pH. where prot’onation of His232 by t’he hydrate might be rate limiting. The pH dependence of the careatinase react,ion may be dominated by the titration of His232. i$“r not,e. however, that the tetrahedral adduct is observed onlv with t,he inhibitor CMS, which is not hydrolyzed. ?Gomeasurements are available of a creat’ine complex at pH 5 or 6. The t,etrahedral adducts of these ligands differ in their

--_-

charges. which may discriminate subst,ratcti and inhibitors. All substrates of creatinase known are positively charged guanidinium or arnidinium groups (Robert,s & Walker. 1985). A further comment seems appropriate c*oncrrrririg the fact that we were able to t,rap and a,nalyzr a t,rur enzyme--subst,rat’e complex. This is likely t)o br due to several fac+ors. The enzyme is relatively slow (k,,, = 0.246 s ‘: Yoshimoto et al.. 1976) and carystallizes rapid (overnight) at coltl-room temperature. where turnover is further reduced. Morr important. perhaps. is the fact t,hat. the ligands are t,rapprd in the enzyme and seemingly unable to escapewithout at least, m&ion of thr side-chains of Arg64 and Arg33.5. Wr even suspect’ that the rnzvme ma? undergo domain motions for subst’rat’e upta,ke and product release led by the typical domain-wise, construction of the molecule found in ot,htr proteins known to undergo hinged domain motion (Kennet t & Huber. 1984). An appropriate experiment woultl bt~ to anal!;z/.r crpstals of t’he f’rrc enzyme, which we’ did. We discovered, however. that’it has the ubiyuitous inhibitar bicarbonat,e bound. which we identified in this way (seeabove).

The active sitct of t)he enzyme is a poc+krt deepI>. buried in the moleculr. Arg335 and Arg64*t block the entrance of the acative site pocket, Substrate binding and product releasereyuires a displacement of one or both of these residues. which is possible associated with a domain motion. Each monomer of the enzyme ha,s two clearly defined domains (Fig. 11). The small X-terminal domain runs from residue I to residue 160. The large domain runs from residue 161 to residue 402. Each of’ t~hr~two ac+tivrh sites of t)he dimeric enzyme is made, by rc~sidursof the large domain of ant monomer and some r&dues of t,he small domain of thr other monomr~r. (:ompariny the structure of the monoc*linicachrvstal a,nd thr trigonal crystal. we ha,vr obsc,rved a verb small domain motion. Figure II shows the two struct,urrs superimposed by fit’tiny thta two large domains. It c1a.nb(x se’~n t.hat thus small domains deviate t,y rotations of’ about 1‘. ‘l’hc>rexis a hinge around residues f 55 to 160 wherfs t hf. t.wo domains are connected. The motion is vchrv small and it does not affect appreciably t hcbposit’ion of’ thcx rrsidu~s in the active sit’e. The ta’o struc+urrs arf‘ calosedfi)rms. but the small motion might indicat’cl thckdirec%ionof’ a larger motion during cbatalysis. A domain mot ion of that sort would displace Arg64*. which blocks tAc> entranc*t~of the activta site, and Phe6%*. which hold:: the (~‘C2) methv1 group. Alt,erna~tivel~. t.hr displacbchment of A4rg64* and Phe6%* vould he ac~hi~,vrtl1)~a tra,nslation and/or a rot.ation of on(’ subunit M:itfi respect to t’hr ottrt,r.

t The asterisk denotesthat, t,hp residuehrlongs to thtl other subunit of thr dimrr.

Enzymatic

Mechanism

609

of Creatinase

ARG

---4-m&272

ASP

Figure 11. Stereo (” plot of 2 superimposed structures of the enzyme creatinase in the monoclinic and the trigonal forms. Two clearly defined domains can be seen. A small x-terminal domain is visible at the top of the Figure. A large C-terminal domain occupies the bottom of the diagram. The least-squares fit of the 2 structures was done using only the * main-chain atmoms of the large domain. The small domains are slightly rotated with respect to each other. The local a-fold axis relating the 2 monomers of creatinase (which is a crystallographic P-fold axis in the trigonal crystal form) is shown as a straight line

Tt is int,eresting that the alkylation of Cys298 inactivates the enzyme (Egloff, 1990). Cys298 is far from the actJive site. It is possible that its alkylation prevents the domain motion (or the subunit motion) and therefore blocks the enzyme in one of the forms, either open or closed. A crystallographic analysis of the alkylated enzyme is planned. We thank D. Turk for assistance with the program MAIN, R. Engh with the program XPLOR and M. Schneider for help with some calculations. Professor P. Bartlett made valuable suggestions on the enzyme mechanism. The support of the Deutsche Forschungsgemeinschaft (grant, HUl69/10-1) is gratefully acsknowledgrd.

References Appleyard. (:. & Woods. D. D. (1956). J. Gen. Microbial. 14. 351-365. Bennett. W. S. & Huber, R. (1984). CR8 Crit. Rev. Biochem. 15, 291-384. Briinger. A. T., Kuriyan. J. & Karplus, M. (1987). 8cie?zce, 235, 458-460. (‘olman. I’. M.. Fehlhammer, H. & Bartels, K. (1975). In Prystalloyraphic Prague lVchoo1

Computing

Techniques:

(Ahmed. F. R., eds). pp. 248-258.

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Huml, K. & Munksgaard,

Sedlacek. B.. Copenhagen. (‘rowther, R. A. bt Rlow. D. M. (1967). Acta Crystallogr. 23, 544-548. Egloff, K. (1990). Ph.D. thesis, Ludwig-MaximiliansCniversitat, Miinchen. Fehlhammer, H. & Bode, W. (1975). J. Mol. Biol. 98, 683-692.

Hoemen, H. W., Knof, S. H., Bartlett, P. A.. Huber, R.. Moellering, H. & Schumacher. G. (1988). J. Mol. Biol. 204. 4177433. Hoppe, W. (1957). Acta Crystallogr. 10. 750-751. Huber, R. (1965). Acta Crystallogr. sect. A, 19. 353-356. Huber. R. & Kopfmann, G. (1969). Acta C’rystallogr. sect. A, 25, 1433152. Huber, R. & Schneider, M. (1985). J. Appl. (‘rystalloyr. 18. 165-169. Huber, R.. Schneider. M., Epp. 0.. Mayr, I.. Messerschmidt, A., Pflugrath, J. & Kayser. H. (1987). J. Mol. Biol. 195, 423-434. International Tables for Crystallography, vol. A (Hahn, T., ed.), D. Reidel, Dordrecht Holland. Jack, A. & Levitt, M. (1978). Acta Crystallogr. srct. .4, 34. 931-935. ,Jensen; L. H. (1955). Acta Crystallogr. 8. 237-240. .Jones, T. A. (1978). J. Appl, Crystallogr. 11, 268-272. Lattman. E. E. (1985). Methods Enzymol. 115. 55-57. Matsuda, Y., Wakamatsu, N.. Inouye, Y., Uede. S., Hashimoto, Y., Asano, K. & xakumara. S. (1986). Chem. Pharm. Bull. 34. 2155-2160. Mendel. H. & Hodgkin, D. C. (1954). Acta Crystallogr. 7, 443-446. Messerschmidt. A., Schneider, M. & Huber. R. (1990). J. Appl. Prystallogr. in the press. Miyoshi, K.. Taira, A., Yoshida, K.. Tamura, K. & Uga, S. (1980a). Proc. Japan Acad. 56(B). 95-98. Miyoshi. K.. Taira, A., Yoshida, K.. Tamura, K. & Uga, W. (1980b). Proc. Japan Acad. 56(B), 99-101. Pflugrath, J. 8: Messerschmidt, A. (1987). ,I. Appl. Crystallogr.

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Remington, S. J., Wiegand, G. & Huber. R. (1982). .I. il/lol. Riol. 158, 111-152. Roberts. J. ?J. & Walker. J. B. (1985). J. Hiol. Chem. 260, 13502-13508. Rossmann, M. G. & Blow, D. M. (1962). Actn Crystallogr. 15, 24-31.

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Roche. J.. Lacombe, G. & Girard. H. (1950). Hiochim. Biophys. Acta, 6. 210-216. Shimizu, F.. Kim, CJ. M., Shinmon. Y. & Yamada. H. (1986). Arch. Microhiol. 145, 322-328. Edited

Beigemann. II’. (1974). Ph.D. thesis. Techniwht Univrrsitlt, Miinchen. Yoshimoto. T., Oka. 1. & Tsuru. I). (197ti). Arch. Biochrn~. Biophys. 177. 508-5 1.5.

by H. IV. Matth,ews