E L S E V I E R Journal of Molecular Structure 442 (1998) 11 - 17

Journa l of

MOLECULAR STRUCTURE

Computational design of biomimetic compounds: urease an example

Csilla Csiki, Marc Zimmer*

Chemistry Department, Connecticut College, New London, CT 06320, USA

Received 5 December 1996; accepted 17 February 1997

Abstract

A combination of database, molecular mechanical and molecular dynamical analyses has been used to find/design a series of structural urease model systems. Although the crystal structure ofurease is known, the solid state structure does not contain a urea substrate molecule and a lot still needs to be learnt about the coordination of urea to nickel. The crystal structure ofurease and the proposed mechanism of its action were used to define the parameters for a model compound. The ideas used to design the urease mimic could be used to design many other biomimetic systems. © 1998 Elsevier Science B.V.

Keywords: Computational design; Biomimetic compounds; Urease; Molecular mechanics

1. Introduct ion

Structural and functional biomimetic compounds are frequently synthesized to model the active site o f a protein [1]. Choosing a iigand to achieve some desired structure in a bioinorganic model compound is largely a matter o f trial and error. Currently, many efforts are being undertaken to make this process more efficient, and the most promising advances are in the field o f combinatorial chemistry [2]. We will present a computational method for the rational o f design model compounds with specific structural properties, and we will show how the method has been used to propose a series o f potential urease mimics.

The first step in designing a model system is to establish a list o f properties the model is to mimic. In order to prevent duplicating published work and to use as much o f the available structural data as

* Corresponding author.

possible, the Cambridge Structural Database (CSD) [3] is searched for any structures that have properties similar or identical to those desired for the mimic. The CSD is used because it is a vastly underutil ized resource that contains more than 150000 crystal structures, and can easily and efficiently be searched for structural motifs.

The 'bu i ld ing ' modules o f model ing programs are used to patch together the structural units obtained in the CSD search or to modify the iigands so that they have the desired properties before using molecular mechanical methods to confirm that the designed complexes are sterically feasible. Molecular mechanics (MM) have often been used to analyze bio-inorganic systems [4], however they have not been used to design model systems. I f specific electronic properties are required, quantum mechanical methods can be used to screen potential models for those having the most desired properties. In the remainder o f this paper we will show how this method has been used to design a family o f urease model systems.

0022-2860/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved PI1 S0022-2 860(97)00089-6

12 C. Csiki, M. Zimmer/Journal o/'Molecular Structure 442 (1998) 11-17

H20

I

Ni2

H2 N__.._..~! NH2

/ Nli I

~" N!2 OH 2 Nil ......

• O,.,H

\ \

H2N~.~_C/NH2

H A

N i l

\ ~ Urea

/

/

Fig. 1. Proposed mechanism for the urease catalyzed hydrolysis of urea.

Urease was the first enzyme that was ever crystal- lized [5] and for many years textbooks have used urease as an example that a protein can be an enzyme without containing an organic coenzyme or a metal ion. However, in 1975 it was found that urease is one of only four enzymes that contains nickel [6]. It con- tains two nickel ions that are presumed to be the active site for the hydrolysis o f urea catalyzed by urease. Nearly 70 years after the first urease crystals were grown the crystal structure of urease was solved [7], as were the structure o f the apoenzyme, two active site mutants [8] and a mononickel mutant [9]. The two nickels in urease are 3.5 A apart and are bridged by a carbamylated lysine. One nickel has a very unusual three coordinate geometry (pseudotetrahedral) while the other is five coordinate.

Urease is an important component o f the nitrogen metabolism in plants [ 10] and microbes [ 11 ]. It cataly- zes the hydrolysis o f urea to form ammonia about 1014 times faster than the uncatalyzed reaction. It has been implicated as a virulence factor in various pathogens

[12], and agriculturally the rapid hydrolysis of fer- tilizer urea by soil bacteria results in alkaline induced plant damage and inefficient nitrogen utilization. The initial mechanism [ 13] proposed for the hydrolysis of urea by urease has been modified [14], as shown in Fig. 1, to accommodate new information obtained from the crystal structure.

None of the crystal structures of urease contain urea and many questions about the mechanism of urease catalyzed hydrolysis remain unanswered. Therefore we have attempted to rationally design a urease model system.

1.1. D e s i r e d p r o p e r t i e s f o r the urease m o d e l s y s t em

Computational methods were used to design a model system that would, ideally, be easy to synthe- size, contain two nickel ions and have a cavity between the two metal ions that is large enough to contain urea bound in the manner shown in Fig. 1. The compound would model the structure of the

C. Csiki. M. Zimmer/Journal of Molecular Structure 442 (1998) 11-17 13

Ni2

..

> 2.ooA

i H2

H 2 N ~ .~

120 °

Nil Fig. 2. The intermediate structure for which we are trying to design a model. The dihedral angle between the unhybridized p orbital on the urea carbon and the lone pair of the hydroxyl oxygen was rotated to find the minimum and maximum Ni-Ni distances for the model.

intermediate and not necessari ly the reactivity o f urease.

The solid state structure o f urease has a n i cke l - nickel distance of 3.5 A.. However, this structure does not contain substrate urea and it is possible that coordination o f urea by Ni l can result in a signifi- cantly different N i - N i distance for the catalytically active intermediate, especial ly i f lys-217 no longer bridges the two nickels upon substrate binding. Two approaches can be taken to model ing the active site. A bridging ligand can be used to keep the two nickels about 3.5 ,A, apart, or a more rigid ligand system can be used that will hold the two metal ions in such a way that the mechanism shown in Fig. 1 can occur. We have chosen the latter approach, and have designed a model system that will hopefully be a structural mimic o f the intermediate shown in the box in Fig. 1.

The minimum and maximum N i - N i distances desirable for a model system were determined by drawing an ideal intermediate as shown in Fig. 2. Standard bond lengths and angles were used wherever possible with a non-bonded distance o f 2.00 A, between the urea carbon and the oxygen that is coor- dinated to Ni2. Since the hydroxyl lone pair has to be perpendicular to the plane o f the urea molecule the only variable is the dihedral angle shown in Fig. 2. Monitoring the N i - N i distance while rotating this dihedral angle reveals that the distance should be

between 3.16 A and 5.36 .~ i f the ideal geometry shown in Fig. 2 is assumed.

The urease mimic should have a ligand framework that will hold the two nickel ions at the calculated distance, and coordinate the metals through oxygens and nitrogens, as these make up the coordination sphere o f the metals in the enzyme. These criteria are shown diagrammatical ly in Fig. 3 and were the basis o f the CSD search. In order to ensure that all the ligands that would meet the requirements for the model system were found we searched the database for all the transition metal complexes fulfilling the criteria. The reasoning behind this decision was that it should be relat ively simple to take a ligand system that met all the requirements for a urease model sys- tem, but had the wrong metals, and synthesize it with nickel.

2. Experimental

The Cambridge Structure Database (CSD) [3] V5.11 was searched for the fragment shown in Fig. 3. Version 5.11 of the CSD was released in

N/O,,~ .......................... N/O~

/0". .... , ,,N/O~ ~ "

Fig. 3. Structural requirements for the urease model system used in the CSD search.

14 C. Csiki, M. Zimmer/Journal o[" Molecular Structure 442 (1998) 11 - 17

Fig. 4. Structure of M 2L l showing the cavity that can be occupied by urease.

April 1996 and contains 152 464 crystal structures. All the hits were stored and converted to MacroModel format using the csdconv program.

MacroModel v. 5.5 was used for all the molecular mechanical analysis. The MM2* parameter set was used with Hancock et al.'s [15] nickel parameters. As is usually the case in inorganic molecular mechanics the torsions around the metal ion were taken as zero. Molecular dynamics simulations were used to examine the flexibility of the nickel com- plexes. Five-hundred-ps simulations with 1.5-fs time- steps at 500 K were sampled 500 times. Dihedral Monte Carlo searches [16] were undertaken in which all rotatable bonds were varied. During the search procedure minimization continued until con- vergence was reached, or until 1500 iterations had been performed. Minimization occurred 'in vacuo' and a derivative convergence criterion of 0.05 kJ mol -I was used. Structures within 50 kJ mo1-1 of the lowest energy minimum were kept and a usage directed method [17] was used to select structures for subsequent MC steps.

3. Results

3.1. Finding a model compound

Ninety hits were obtained for the substructure shown in Fig. 3 with a metal to metal distance of between 3.00 and 5.80 A.. A slightly larger distance than that predicted on the basis of Fig. 2 was used in the CSD search in order to find binuclear complexes

with larger metal ions that could be replaced with nickel, resulting in model compounds with the desired metal-metal distances. Forty % of the hits were cop- per complexes and 15% contained nickel. Amongst the structures obtained from the CSD search was a family of compounds that fulfilled all our requirements. They all had the substituted macrocycle 1,4,8,11- tetrakis(2-pyridylmethyl)- 1,4, 8, 1 l-tetraazacyclotetra- decane (L l) as a ligand holding the metal ions in place. The ligand is a potential urease structural model because the metal ions are held apart in place by a fairly flexible framework that allows the metals to be between 3 and 6 A., it has an empty cavity between the metals which could accommodate urea (Fig. 4; see also Fig. 7), it can be synthesized in two steps [18] and is easily modified (Fig. 5). Five bicop- per complexes with L 1 were found with Cu-Cu dis- tances of between 3.993 and 5.741 ,~. The CSD codes for the hits were DUJKEY [18], VEGKOH [19], VEGKUN [19], VEPPUB [20] and VEPRAJ [20]. In DUJKEY the copper ions are 5.741 ,~ apart, they are five coordinate and there are no bridging ligands between the copper ions. In VEGKOH the coppers are octahedral, 3.714 A apart and bridged by a hydroxy group. VEGKUN has t~jnitrato bridged octahedral cop- pers which are 4.651 A apart, VEPPUB has tx-fluoro bridged octahedral coppers which are 3.993 .& apart and VEPRAJ has octahedral ~-chloro bridged octa- hedral coppers which are 4.468 /k apart. No nickel complexes of the ligand were found; however, there is no reason why nickel should not bind to the ligand in a similar fashion to copper.

C. Csiki, M. Zimmer/Journal of Molecular Structure 442 (1998) 11-17 15

F1 R

Fig. 5. The structure of 1,4,8,11-tetrakis(2-pyridylmethyl)-l,4,8,1 modified by varying n and R.

In order to establish whether the ligand L ] forms any other complexes that do not fall within the param- eters defined in the CSD search, we searched the data- base for L 1 and found that the solid state structures of three other complexes [21] with this ligand are known. The complexes were not found in the original search because they were not bimetallic complexes or because the meta l -meta l distance was greater than 5.8 A. While all the complexes discussed in the previous section were copper(II) complexes, SEWNEN has two tricoordinate copper(l) ions that are 6.74 ,~ apart [22]. Although the copper complexes with the

l-tetraazacyclotetradecane (n = 1 and R = H). The ligand can easily be

L 1 are bimetallic, mononuclear rhenium [23] and ruthenium [24] complexes were found. Presumably this is due to the large size of the two second row transition metals. Nickel is similar in size to copper and is more likely to adopt bimetallic complexes.

3.2. M o l e c u l a r m e c h a n i c a l ana lys i s

The copper ions in the solid state structure of VEPRAJ were replaced with nickels, the bridging /x-chloro ligand was removed and the structure was minimized. A molecular dynamics simulation of the

5 . 4 0

A

t r o

m s

4 . 4 0 1 100'

I ~ c t u r e Number 300' 400' 501

Fig. 6. A plot of the Ni Ni distance as a function of structure number. Structures were saved every picosecond during a 500-ps molecular dynamics simulation at 500 K.

16 C. Csiki, M. ZimmerJournal (~[' Molecular Structure 442 (1998) 11-17

Ni-l~i d i s t a n c e

Fig. 7. An overlap of all the low energy conformations of Ni2L t within 25 kJ mol ~ of the lowest energy conformation.

binickel complex was run to ensure that the cavity between the metal ions does not collapse and that there is room for urea coordination. The nickel-nickel distance was monitored as a function of time and is shown in Fig. 6. The plot shows that although the ligand is fairly flexible, that is the N i - N i

distance varies from 4.40 to 5.40 ~, without under- going a major conformational change, it does not collapse.

Another possible shortcoming of the structural model could be that it adopts a low energy conforma- tion that is not a viable structural urease model, and

c. Csiki, M. Zimmer/Journal o f Molecular Structure 442 (1998) 11-17 17

model could be that it adopts a low energy conforma-

tion that is not a v iable structural urease model , and

that this conformat ion was not observed in the

molecu la r dynamics s imulat ion because it is

separated f rom the nickel 'mu ta t ed ' V E P R A J

structure by a large energy hill. In order to ensure

that no other low energy conformat ions exist we

under took a Monte Carlo dihedral conformat ional

search o f the complex . Fig. 7 shows the over lap

o f all the conformat ions that were found within

25 kJ mol -~ o f the lowest energy conformat ion. All

these low energy conformat ions are v iable urease

mimics .

Molecu la r mechan ica l analysis o f the binickel

complexes formed with substi tuted macrocyc les

1,4,8,1 1- te t rakis(2-pyr idylmethyl)- 1,4, 8,11 -tetraaza-

cyc lopen tadecane and 1,4,8,1 1-tetrakis(2-pyridyl-

methyl ) - 1,4, 8,1 1- te t raazacyclohexadecane , revea led

that the N i - N i dis tance was not substant ial ly changed

by replac ing the five m e m b e r e d rings with six m e m -

bered rings in the macrocyc le . However , the width o f

the cavi ty was decreased, this is not surprising con-

s ider ing the smal ler bite size o f p ropanediamine vs

e thy lened iamine [25].

4. Conclusion

The proposed m e c h a n i s m for the hydrolysis o f urea

by urease was used to establish structural parameters

for a urease mode l system. A combina t ion o f database

and molecu la r mechan ica l analysis led to the

d i scovery o f a l igand f ramework that would form a

b inuclear nickel comp lex that might be capable o f

comp lex ing urea. The me thod used to design/f ind

the urease m i m i c k i n g l igand can in theory be appl ied

to f inding mode l compounds for any b io logica l

systems.

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