Catalytic Mechanism of Class A b -Lactamase

7/31/2019 Catalytic Mechanism of Class A b -Lactamase

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b-Lactamases are produced by pathogenic bacteria andcontribute to a resistance to b-lactam antibiotics by deterio-rating the pharmacological effect of catalyzing the hydrolysisof the b-lactam ring.1) b-Lactam antibiotics react with b-lac-tamase in the same way as penicillin-binding protein (PBP);

i.e., an acylation reaction is initiated by the opening reactionof the b-lactam ring. In the case ofb-lactamase, however, thedeacylation reaction occurs after the acylation reaction andits activity is recovered, which differs from PBP. We investi-gated the deacylation reaction in order to elucidate the cat-alytic mechanism of class A b-lactamase by quantum chemi-cal calculations.

Since the report by Knox et al. suggested that the E166Amutant of b-lactamase from Bacillus licheniformis inducesthe accumulation of an acyl-enzyme intermediate,2) the cat-alytic residue involved in the deacylation reaction has beenpresumed to be only Glu166. However, it is difficult to con-clude that the deacylation reaction occurs only due to Glu166

because the distance between Ser70Og and Glu166Oe is toofar to interact (ca. 3.74 ) according to the results of ourmolecular dynamics (MD) study on the structure ofb-lactamantibioticsb-lactamase complex (Fig. 1). It is thought thatnot only Glu166 but also Lys73 is involved in the deacylationreaction because Lys73Nz is located between Ser70 andGlu166 and interacts with Ser70Og and Glu166Oe, nearlyforming hydrogen bonds (2.77 and 3.21, respectively). Fur-thermore, it is thought that a hydrogen bond network amongLys73Nz, Ser130Og and the carboxyl group of the substrate(b-lactam antibiotics) should be taken into account. Ishiguroet al. also discussed the importance of the hydrogen bondnetwork based on the results of their molecular mechanics

calculations.3)

In the present study, we investigated the tetra-hedral intermediate formation process, which is the first stepof the deacylation reaction by b-lactamase and is caused by

the interaction between a water molecule and b-lactam an-tibiotics (benzyl penicillin)b-lactamase complex, by meansof the ab initio molecular orbital (MO) method, and we clari-fied the activation energy using the structure and potentialenergy changes for the reaction and the function of the hy-

drogen bond network binding by Glu166OeLys73NzSer70OgSer130Ogthe carboxyl group of benzyl penicillin.

April 2000 Chem. Pharm. Bull. 48(4) 447453 (2000) 447

To whom correspondence should be addressed. 2000 Pharmaceutical Society of Japan

Catalytic Mechanism of Class A b-Lactamase. I. The Role of Glu166and Ser130 in the Deacylation Reaction

Masayuki HATA,* Yasuyuki FUJII, Miho ISHII, Tyuji HOSHINO, and Minoru TSUDA

Laboratory of Physical Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Chiba 2638522, Japan.

Received April 30, 1999; accepted December 10, 1999

The tetrahedral intermediate formation process, which is the first step in the deacylation reaction by class A

b-lactamase, was investigated by the ab initio molecular orbital method. In this study, benzyl penicillin was usedas the substrate. From the results of our molecular dynamics study of the structure of b-lactam antibioticsb-lac-tamase complex, the substrate, Ser70, Lys73, Ser130, Glu166 and a water molecule for the deacylation reaction

were considered for construction of a model for calculation. The calculation results indicated that Glu166 plays a

role in holding a water molecule, which is necessary for the deacylation reaction, and that the hydrogen bond

network among Lys73Nz, Ser130Og, and the carboxyl group of the b-lactam antibiotics was formed by the up-take ofb-lactam antibiotics by b-lactamase. The activation energy for this reaction was 33.3 kcal/mol, and it isvery likely that the reaction occurred at body temperature. Subsequent calculation results obtained by using the

model excluding Ser130 and the carboxyl group of the substrate indicated that the activation energy for this re-

action was 40.8 kcal/mol, which is 7.5 kcal/mol higher than that of the previous reaction. It was found that the hy-

drogen bond network plays an important role in decreasing the activation energy for the tetrahedral intermedi-ate formation reaction. Lys73Nz, which is located at the edge of the hydrogen bond network, played a role informing a hydrogen bond with Glu166Oe in order to help the deacylation reaction. The role of amino acidresidues around the active site of class A b-lactamase was also discussed.

Key words b-lactamase; tetrahedral intermediate; ab initio molecular orbital method; hydrogen bond network; Lys73; deacyla-tion reaction

Fig. 1. Active Site of Benzyl Penicillin-Bound Acyl-Enzyme IntermediateObtained by MD Calculation

Important residues for the deacylation reaction by b-lactamase are extracted and areshown in this figure. Numerals are distance between each atom in . WAT is a watermolecule for the deacylation reaction by class A b-lactamase.


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mase. The oxygen atom of the water molecule held byGlu166Oe in the ES complex (a) formed a covalent bond

with the carbonyl carbon of the b-lactam ring bound withSer70Og (1.47 ) and a hydrogen bond with Glu166Oe(2.75 ). The hydrogen bond chain Lys73NzSer130Ogthecarboxyl oxygen atom of the substrate, was always main-tained in the tetrahedral intermediate formation process.

Water Molecule-Holding Mechanism in the Deacyla-

tion Reaction by b-Lactamase In Fig. 4(a), proton migra-tions from Lys73Nz to Ser130Og and from Ser130Og to thecarboxyl oxygen of b-lactam antibiotics occurred. Thischange will be described in Discussion. When the water mol-ecule held by Glu166Oe approached the carbonyl carbon ofthe b-lactam ring bound with Ser70Og, the structure shownin Fig. 3(b) appeared due to rotation of the water molecule

and the carboxyl group of Glu166. The distance between theoxygen atom of the water molecule and the carbonyl carbonof the b-lactam ring was 2.70 . It should be noted that a hy-drogen bond is formed between Glu166Oe and Lys73Nz instructure (b). This hydrogen bond plays a role in stabilizingthe water molecule which is held by Glu166 and is used forthe deacylation reaction. When the water molecule ap-proaches the carbonyl carbon of the b-lactam ring boundwith Ser70Og, it begins to decompose into a proton andOH. Hwa, a proton in the water molecule, migrates toGlu166Oe, while OH approaches the carbonyl carbon ofthe substrate, thereby leading to the formation of a TS struc-ture (c). The distance between the proton Hwa and Glu166Oe

is 1.04 , which is almost equal to the bond length of theOH bond, and the distance between Ow of OH and thecarbonyl carbon Cs of the b-lactam ring of the substrate is

1.82 . The distance between Cs and Ser70Og increasesgradually from 1.34 in structure (a) and becomes 1.40 in

structure (c). It was clearly indicated in the vibrational modeshown in structure (c) (Fig. 3), that the proton from the watermolecule which was involved in the deacylation reaction byclass A b-lactamase connects with Glu166Oe, and OH con-nects with the acyl carbon of the b-lactam ring of the sub-strate. Figure 2 shows that, starting from the ES complexstructure (a), the reaction intermediate (d) was formed viastructure (b) and the TS structure (c); i.e., (a) was connectedwith (d) by the steepest path. It was found from the potentialenergy change shown in Fig. 2, that the activation energy forthe tetrahedral intermediate formation reaction was33.3 kcal/mol. It is very likely that this reaction occurs atbody temperature. The role of Glu166 is to hold a water mol-

ecule for the deacylation reaction by class A b-lactamase inthe reaction system. It is considered that an acyl intermediateaccumulates in the Glu166Ala mutant ofb-lactamase,2) be-cause the mutant loses this function.

Discussion

Reaction mechanism There have been many studies onmechanism of substrate inactivation by b-lactamase. Stry-nadka et al. reported from the result of their X-ray crystallo-graphic analysis of RTEM-1 b-lactamase, that Lys73Nz isneutral because the pKa of Lys73 decreases due to a positiveenvironment that is produced by residues around Lys73, thatLys73 only takes part in the acylation of the OH group of

Ser70, and that only Glu166 takes part in the deacylation re-action ofb-lactamase.17) On the other hand, Damblon et al.showed from their NMR experiments that only Glu166, not

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Fig. 4. Structural Changes Following the Tetrahedral Intermediate Formation Reaction in the Deacylation Step by Class A b-Lactamase

(a)(d) correspond to that shown in Fig. 2


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the previous section, it was stated that a hydrogen bond net-work is produced between Lys73, Ser130 and the carboxylgroup of the b-lactam antibiotics, and that the proton relaybetween the hydrogen bond network is involved in the activ-ity of class A b-lactamase. When the substrate, benzyl peni-cillin, is caught by b-lactamase, its carboxyl group is incor-porated in the hydrogen bond network. The protonatedLys73Nz is neutralized and is activated by the proton relayvia Ser130Og. The mechanism in which the hydrogen bondnetwork works effectively is shown in Fig. 4. The mechanismshown in this figure is reasonable because the potential en-ergy of the structure, which assumes that the tetrahedral in-termediate is produced without proton relay from Lys73Nzto the carboxyl group of the substrate via Ser130Og, is23.5 kcal/mol higher than that of structure (d).

Ser130 and the carboxyl group of the substrate were ex-cluded from the model reaction system to estimate the effectof the Lys73Ser130substrate hydrogen bond network. The

potential energy change and the structural changes obtainedby quantum chemical calculations are shown in Figs. 5 to 7,respectively. A hydrogen atom of protonated Lys73Nz, whichforms a hydrogen bond with Ser130Og, was removed fromthis model structure because Ser130 was not included in themodel structure. In Fig. 7, structural changes from an initialstructure (A) to a TS structure (D) did not differ greatly fromthat in Fig. 4. However, it is observed in the only normal vi-brational mode with an imaginary frequency of the TS struc-ture (D) that proton Hwa from the water molecule involvedin the deacylation reaction is oriented toward Lys73Nz (Fig.6). Moreover, after passing the TS structure (D), a point ofinflection appeared in the potential energy change shown inFig. 5, which did not appear in that shown in Fig. 4. Thestructure is shown in Fig. 7(E). Structure (E) is a point of in-flection that appeared as a result of stabilization in which theproton Hwa from the water molecule changes its moving di-rection naturally in order to break the hydrogen bond with

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Fig. 8. Structures of Expanded ModelsAtoms which are also used for the elucidation of a tetrahedral intermediate formation reaction mechanism (Results) are shown by circles. In Results, atoms marked by asterisks

in these figures were always fixed. Residue shown by a thick line is benzyl penicillin.


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the Ow oxygen atom of the water molecule and to form a hy-drogen bond with Lys73Nz. From structure (E), the ex-change of the hydrogen bond occurred and a stable tetrahe-dral intermediate (F) was produced; i.e., in the tetrahedral in-termediate structure (F), Hz, which connects with Lys73Nz,forms a hydrogen bond only with Ser70Og (1.83), whereasin the TS structure (D), it interacts with Glu166Oe andSer70Og, whose distances are 2.49 and 2.32 , respectively.It is thought that an exclusion of Ser130 and the carboxylgroup of the b-lactam antibiotics causes these structuralchanges. Due to this exclusion, the activation energy of thisreaction is 40.8 kcal/mol, which is 7.5kcal/mol higher thanthat obtained in Results. For this reason, it is concluded thatthe hydrogen bond network consisting of Lys73, Ser130 andthe carboxyl group of the b-lactam antibiotics plays an im-portant role in remarkably decreasing the activation energyof the tetrahedral intermediate formation reaction, which isthe first step of the deacylation reaction, by a proton relayamong this network.

Role of Amino Acid Residues around the Active Site of

Class A b-Lactamase It is considered that amino acidresidues and solvent molecules, which are located around theactive site, also affect enzymatic reaction mechanisms andthe potential energy change. Though it is quantitatively diffi-cult to take all these factors into computation, we constructeda larger model than that shown in Fig. 4, based on the struc-ture shown in Fig. 1. The model is called expanded model.The expanded model includes four active site residues(Ser70, Lys73, Ser130, Glu166), six residues near the active-site (Ala69, Thr71, Asn132, Ser235, Gly236, Gln237) andthe full structure of the benzyl penicilloyl moiety. The watermolecule which is involved in the expanded model as a sol-vent molecule is only WAT, as shown in Fig. 1, because thenearest water molecule from WAT did not interact with theactive site, including WAT.

From this structure, the positions of the atoms included inboth the expanded model and the model reaction system inFig. 4 were changed into those of structure (a) to (d) in Fig.4. (Because atoms marked by asterisks in Fig. 4(a) were fixedthrough investigation on the deacylation reaction mechanismof class A b-lactamase, the positions of each atom are equalto those shown by asterisks in Fig. 8.) The structures areshown in Fig. 8.

An expanded model for an acyl-enzyme intermediate wasalso constructed. First, an acyl-enzyme intermediate (struc-

ture (Int.)) was optimized by the same method as describedin Method. The geometry was almost the same as that shownin Fig. 1. Second, the optimized data was put through thesame process, converting (a) to (d). The structure (structure(Int.)) is shown in Fig. 9. For these expanded models, onlypotential energy calculations were performed at the HF levelusing a 6-31G** basis set because of computational limits.

The result is shown in Fig. 10. It should be noted thatstructure (Int) is more stable than structure (a), although thepotential energy of structure (Int.) is 6.4 kcal/mol higher thanthat of structure (a). This result suggests that the amino acidresidues around the active site of class A b-lactamase mightplay an important role in the stabilization of protonated

Lys73 and the carboxyl group of the substrate in acyl-en-zyme intermediate. It is predicted that structure (Int) links tostructure (a) by a proton relay from Lys73Nz to the carboxyl

group of the substrate via Ser130Og, on the potential energyhypersurface. The mechanism is now being investigated.

The activation energy for tetrahedral intermediate forma-tion process is 19.9 kcal/mol ((a)(c)). The value issmaller than that shown in Fig. 2 (33.3kcal/mol). Further-more, the stabilization energy from the TS structure (c) to

the tetrahedral intermediate (d

) is 13.9 kcal/mol. The valueis larger than that shown in Fig. 2 (5.2 kcal/mol, (c)(d)). Itis thought that the amino acid residues around the active-siteof class A b-lactamase might also play roles in decreasingthe activation energy for tetrahedral intermediate formationreaction and in stabilizing the tetrahedral intermediate.

Conclusions

The conclusions obtained in this investigation are as fol-lows. 1. Glu166 plays a role in holding a water molecule,which is necessary for the deacylation reaction by class A b-lactamase. For this reason, an acyl intermediate accumulatesin the Glu166Ala mutant. 2. The hydrogen bond network

consisting of Lys73Nz, Ser130Og and the carboxyl group ofthe b-lactam antibiotics is formed by the uptake ofb-lactamantibiotics by b-lactamase, and plays an important role in de-

452 Vol. 48, No. 4

Fig. 10. Potential Energy Diagram on Structures (Int.) and (a) to (d),Which Were Obtained by Potential Energy Calculations at the HF/6-31G**Level

Fig. 9. Structure of Expanded Model (Int.)

Atoms shown by circles are used for geometry optimization on the structure (Int.)(see text). Residue shown by a thick line is benzyl penicillin. Numerals are inter-atomicdistances () obtained by geometry optimization.


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creasing the activation energy for the tetrahedral intermediateformation reaction, which is the first step of the deacylationreaction, by the proton relay among this network. 3.Lys73Nz, which is located at the edge of the hydrogen net-work, forms a hydrogen bond with Glu166Oe in order tohelp the deacylation reaction by the water molecule held byGlu166. 4. Amino acid residues around the active site ofclass A b-lactamase might play the roles of stabilizing proto-nated Lys73 and the carboxyl group of the substrate in theacyl-enzyme intermediate, in stabilizing the tetrahedral inter-mediate, and in decreasing the activation energy for tetrahe-dral intermediate formation reaction.

Acknowledgment The authors thank the Computer Center of the Insti-tute for Molecular Science, for the use of the IBM SP2 computer. The com-putations were also carried out by the DRIA System at the Faculty of Phar-maceutical Sciences, Chiba University.

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9) Weiner S. J., Kollman P. A., Case D. A., Singh U. C., Ghio C., AlagonaG., Profeta S., Jr., Weiner P., J. Am. Chem. Soc., 106, 765784 (1984).

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926935 (1983).12) Ryckaert J. P., Ciccotti G., Berendsen H. J. C.,J. Comput. Phys., 23,

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14) Futatsugi, et al. performed a 100 ps MD calculation with a time step of1 femto second (fs) by using an MD-engine.13) When the average dis-tances of one hundred continuous sets of coordinates were plotted, thefluctuation of the hydrogen bond distance (below 3 ) was not morethan 0.1 in 1 ps. This result is to be submitted for publication inNa-ture.

15) Herzberg O.,J.Mol.Biol., 217, 701719 (1991).16) Frisch M. J., Trucks G. W., Schlegel H. B., Gill P. M. W., Johnson B.

G., Robb M. A., Cheeseman J. R., Keith T., Petersson G. A., Mont-gomery J. A., Raghavachari K., Al-Laham M. A., Zakrzewski V. G.,

Ortiz J. V., Foresman J. B., Cioslowski J., Stefanov B. B., NanayakkaraA., Challacombe M., Peng C. Y., Ayala P. Y., Chen W., Wong M. W.,Andres J. L., Replogle E. S., Gomperts R., Martin R. L., Fox D. J.,Binkley J. S., Defrees D. J., Baker J., Stewart J. P., Head-Gordon M.,Gonzalez C., Pople J. A., Gaussian 94, RevisionE2, Gaussian Inc.,Pittsburgh, PA, 1995.

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Catalytic Mechanism of Class A b -Lactamase