熊本大学学位論文

熊本大学学位論文

新興薬剤耐性菌が産生するメタロ -β- ラクタマーゼ の加水分解機構の解明と非可逆的阻害剤の開発

2005 山口 佳宏

Probing the hydrolysis mechanism of metallo-β-lactamases and development of their irreversible inhibitors

2005

Yoshihiro Yamaguchi

Probing the mechanism of hydrolysis of metallo-β-lactamases and development of irreversible inhibitors

Yoshihiro Yamaguchi Metallo-β-lactamases (MBLs) are dinuclear Zn(II) metalloenzymes that catalyze the efficient hydrolysis of a wide range of β-lactam antibiotics including carbapenems and extended-spectrum cephalosporins. The emergence of appearance of these antibiotic-resistant bacteria raises a considerable public health concern and awareness regarding the proliferation and treatment of infectious diseases. More than twenty MBLs have been identified in clinical isolates of major gram-negative pathogens. Furthermore, MBLs show little or no susceptibility to clinically used β-lactamase inhibitors such as clavulanic acid, tazobactam, and sulbactam. Although there have been reported several of compounds that inhibit MBLs, at present no clinically useful inhibitors appear to be available.

One of the causes for this is the rate of MBL dissemination and diversity, which prevents the development of ΜΒL inhibitors. These facts clearly highlight the urgency and importance of improving our understanding of MBLs to design either new β-lactam antibiotics with improved activity against bacteria or inhibitors that are capable of extending the utility and lifetime of currently used antibiotics.

Therefore, the aims of this study were to clarify the common hydrolysis mechanism of MBLs using kinetics and X-ray crystallographic studies and to develop mechanism-based irreversible inhibitors for all MBLs and agents for detecting MBL-producing strains with high sensitivity and selectivity.

The new findings obtained in this study are summarized as follows.

1. Knowledge of the pH dependency in hydrolyzing enzymes is indispensable to elucidate

the reaction mechanism because the active sites generally contain acidic or basic

groups and one protonated form of the acid and base is catalytically active. In

pH-jump experiments, IMP-1, a member of the MBL family and named from a

Serratia marcescens isolate collected in Japan in 1991, was inactivated in an acidic

medium by the dissociation of Zn(II) from the holo-enzyme, where ligands

coordinating to Zn(II) ion(s) may be protonated. The pH dependence for the IMP-1

hydrolysis in the presence of large excess of Zn(II) ion revealed that no functional

group having a pK

between pH 5 and 9 was present. These results suggest that the

pK

value of a bridging OH

to two Zn(II) ions (Zn1-OH

-Zn2), which acts as a

nucleophile to the carbonyl carbon on the β-lactam ring, is lower than 5. It is thought

that the positive charges of the two Zn(II) ions are the main cause for lowering the pK

for water as a nucleophile and assist in the deprotonation of the bridging OH

.

2. To probe the role of the well conserved Asp120 in the active site of IMP-1, site-directed mutagenesis was conducted. The two Asp120-mutants, D120(81)A and D120(81)E, in which asparatic acid was replaced by alanine and glutamic acid, were characterized using CD, metal analyses, steady-state kinetics, and X-ray crystallography. In the steady state kinetic experiments, D120(81)A and D120(81)E showed a remarkable reduction in activity, i.e. a decrease in k

by 10

-10

fold and an increase in K

of several fold to 10

-fold. These results suggest that Asp120 in IMP-1 plays important roles in catalysis and substrate recognition. Furthermore, from pre-steady-state kinetic experiments, an anionic intermediate of nitrocefin having an absorption maximum around 650 nm was not observed for the IMP-1 mutants, suggesting that Asp120 in IMP-1 is not a proton donor to the anionic intermediate of nitrocefin. The pH dependence of the hydrolysis for the IMP-1 mutants indicated that Asp120 is not a factor in decreasing the pK

below 5 for the water bridging two Zn(II) ions. The three-dimensional structures of D120(81)E and Glu120(81)E were determined at a resolution of 2.0 and 3.0 Å, respectively. From the X-ray crystallography results, the structure of D120(81)E (the oxygen atom of Glu120(81)OE1 is presumably not coordinated to the bridging OH

or OH

), the oxygen atom of the carboxylate of Asp120 is essential for coordination to the bridging OH

or OH

by a hydrogen bond to achieve catalytic activity. Thus, the critical role of Asp120 is to orientate OH

or OH

, which acts as a nucleophile to the carbonyl carbon on the β-lactam ring.

3. To develop effective inhibitors against IMP-1, two novel MBL inhibitors,

pentafluorophenyl 3-mercaptopropionate (PFMP) and 3-(3-mercaptopropionylsulfanyl)propionic acid pentafluorophenyl ester (MPAP) were

synthesized. These compounds have a thiol group for the coordination to Zn(II)

ion(s), and an activated ester capable of interacting with the Cys 221 or Lys224 residue

in the active site as a nucleophile. From gel-filtration and dialysis experiments, the

treatment of IMP-1 with each inhibitor resulted in a nearly 100% inhibition, indicating

that both inhibitors inhibited IMP-1 very rapidly and irreversibly. The MALDI-TOF

mass spectra of IMP-1 treated with each inhibitor showed an increase in mass,

corresponding to the sum of the mass of the acyl moiety that is removed from the

pentafluorophenol group from the inhibitor and of IMP-1. Moreover, to identify the

site of amino acid attachment and to determine the three-dimensional structure, the

X-ray crystallography of IMP-1 complexed with MPAP was carried out at a resolution of 2.6 Å. X-ray crystallography revealed that the thiol group bridges between the two Zn(II) ions in the active site and the terminal carbonyl group in the acyl moiety covalently bonds to the side chain N

atom of Lys224 to form an amide bond.

4. To devise a fluorometric method for detecting MBL-producing strains rapidly and selectively, a novel fluorescence detecting reagent, N-[2-(5-dimethylamino-naphthalene-1-sulfonylamino)ethyl]-3-mercaptopropionamide

(DansylC2SH) was synthesized. This compound contains a thiol group as a Zn(II) ligand and a dansyl group as a fluorophore and both groups are linked via ethylendiamine as a spacer. To examine the ability of the fluorescent detecting agent for IMP-1, the fluorescence emission spectra of DansylC2SH in the absence or the presence of IMP-1 were measured. When a IMP-1 buffered solution was added to a DansylC2SH solution, fluorescence enhancement was observed, indicating that DansylC2SH binds in the active site, and interacts with Trp64(28) near the active site.

As mentioned above, these findings shed new light on our understanding of the common hydrolysis reaction mechanism for, not only MBLs, but also binuclear Zn(II) hydrolases that have been discovered recently. The inhibition modes of the inhibitors for IMP-1 designed and synthesized in this study were clarified to be irreversible using kinetics, MALDI-TOF mass, and X-ray crystallographic studies. These inhibitors interacted with IMP-1 to form a covalent bond between the acyl group in the inhibitor and the side chain N

atom of Lys224, leading to irreversible inhibition. Thus, the strategy for the design of irreversible inhibitors and X-ray crystal results in this study would help in the design of mechanism-based inhibitors that target, not only all MBLs, but also on other metalloenzymes such as metallo matrix proteases.

Moreover, it is expected that the fluorescence detecting reagent, DansylC2SH, represents a

lead compound for the further development of detecting agents and specific inhibitors with a

high affinity and selectivity for all MBLs.

本論文で使用した略号一覧表

MBL Metallo-β-lactamase

MALDI-TOF MS Matrix assisted laser desorption ionization-time of flight mass spectrometer

MOPS 3-Morpholinopropanesulfonic acid MES 2-Morpholinoethanesulfonic acid

WT Wild-type

CD Circular dichroism

DMSO Dimethyl sulfoxide

Tris Tris(hydroxymethyl)aminomethane

PDB Protein Data Bank

MW Molecular weight

SDA-PAGE Sodium dodecyl sulfate-polyachrylamide gelelectrophoresis PFMP Pentafluorophenyl 3-mercaptopropionate

MPAP 3-(3-Mercaptopropionylsulfanyl) propionic acid pentafluorophenyl ester

TFA Trifluoroacetic acid

3-MPA 3-Mercapropropionic acid

Hepes 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid PCR Polymerase chain reaction

NCCLS National committee for Clinical Laboratory Standards DansylC2SH N-[2-(5-dimethylamino-naphthalene-1-sulfonylamino)ethyl]

-3-mercaptopropionamide

CA Carbonic anhydrase

本論文は学術雑誌に掲載された次の論文を基礎とするものである。

1. Dependence of Hydrolysis of β-lactams with a Zinc(II)-β-Lactamase Produced from Serratia marcescens (IMP-1) on pH and Concentration of Zinc(II): Dissociation of Zn(II) from IMP-1 in Acidic Medium

Masafumi Goto, Hisami Yasuzawa, Toshihiro Higashi, Yoshihiro Yamaguchi, Akiko Kawanami, Shiho Mifune, Hiromasa Mori, Hitoshi Nakayama, Kumiko Harada, and Yoshichika Arakawa

Biol. Pharm. Bull., 26, 9-594 (2003)

2. Detection of Metallo-β-Lactamase (IMP-1) by Fluorescent Probes Having Dansyl and Thiol Groups

Hiromasa Kurosaki, Hisami Yasuzawa, Yoshihiro Yamaguchi, Wanchun Jin, Yoshichika Arakawa, and Masafumi Goto

Org. Biomol. Chem., 1, 17-20 (2003)

3. Probing the Role of Asp120(81) of Metallo-β-Lactamase (IMP-1) by Site-directed Mutagenesis, Kinetic Studies and X-ray Crystallography

Yoshihiro Yamaguchi, Takahiro Kuroki, Hisami Yasuzawa, Toshihiro Higashi, Wanchun Jin, Akiko Kawanami, Yuriko Yamagata, Yoshichika Arakawa, Masafumi Goto, and Hiromasa Kurosaki

J. Biol. Chem., 280, 20824-20832 (2005)

4. Irreversible Inhibition of Metallo-β-Lactamase (IMP-1) by

3-(3-Mercaptopropionylsulfanyl)propionic Acid Pentafluorophenyl Ester

Hiromasa Kurosaki, Yoshihiro Yamaguchi, Toshihiro Higashi, Kimitaka Soga, Satoshi Matsueda, Haruka Yumoto, Shogo Misumi, Yuriko Yamagata, Yoshichika Arakawa, and Masafumi Goto

Angew. Chem. Int. Ed., 44, 3861-3864 (2005)

目次

第

1

···1

第

2

metallo-β-lactamase (IMP-1)

Zn(II)

pH

第

1

···9

第2節 結果

··· 11

2-1

IMP-1

Zn(II)

··· 11

2-2

IMP-1

Zn(II)

···12

2-3 イミペネムに対する加水分解の pH

···14

第

3

···15

第4節 小括

···19

第3章 部 位 特 異 的 変 異 法 、 速 度 論 解 析 、

X

metallo-β-lactamase (IMP-1)

Asp120(81)

···20

第1節 序論

···20

第

2

···22

2-1 Asp-120(81)変異体、D120(81)A

D120(81)E

···22

2-2 IMP-1

···22

2-3 IMP-1

pH

2-4 WT

IMP-1

···26

2-5 D120(81)A

D120(81)E

X

···28

2-6

D120(81)A

····32

第3節 考察

···33

第

4

···39

第

4

3-(3-Mercaptopropionylsulfanyl)propionic acid pentafluorophenyl ester

metallo-β-lactamase (IMP-1)の非可逆的阻害···40

第

1

···40

第2節 結果

···42

2-1

IMP-1

···42

2-2

2-3

···45

2-4

IMP-1

MS)

···45

2-5 MPAP

IMP-1

X

···47

第

3

···50

第4節 小括

···53

第5章 ダ ン シ ル 基 と チ オ ー ル 基 を 有 す る 蛍 光 プ ロ ー ブ に よ る

metallo-β-lactamase

···54

第1節 序論

···54

第

2

···55

2-1 DansylC2SH

···55

2-2 IMP-1

DansylC2SH

IMP-1

2-3 Alubmin

CA

DansylC2SH

IMP-1

···58

2-4 apo IMP-1

DansylC2SH

Zn(II)イオン

···59

2-5

DansylC2SH

IMP-1

···60

2-6 DansylC2SH

IMP-1

···62

第

3

···63

第4節 小括

···65

第6章 総括 ···67

実験の部···71

謝辞

···88

参考文献···90

第

1

人類が

1940

(PBP)

transpeptidase

そこで耐性菌の克服とより広い抗菌スペクトルを求めて、第

2

3

1980

β-

1990

β-ラクタム剤を不活化する酵素、β-lactamase

β-lactamase

class A

D

class A

C

D

serine-β-lactamase

Zn(II)

class B

metallo-β-lactamase (MBL)

2

MBL

1966

Bacillus cereus

1985

Pseudomonas maltophilia

L1

1991

Bacteroides fragilis

CcrA

Aeromonas hydrophilia

CphA

1994

Serratia marcescens

IMP-1

MBL

MBL

20

MBL

さらに

MBL

B3

れている ¹¹。Fig. 1 に代表的な

MBL

subclass

class B β-lactamase (BBL)

numbering

matured

示す¹¹。例えば

IMP-1

Asp120(81)

BBL numbering

120

matured IMP-1

81

Subclass B1 (..HxHxD..)

BcII DVIITHAHADRIGGI CcrA TFIPNHWHGDCTGGL IMP-1 GSISSHFHSDSTGGI VIM-2 RAVSTHFHDDRVGGV BlaB MNIATHSHDDRAGGL IND-1 AVFATHSHDDRAGDL

Subclass B2 (..NxHxD..)

CphA EVINTNYHTDRAGGN

Subclass B3 (..HxHxDH..)

L1 LILLSHAHADHAGPV FEZ-1 ILLISHAHFDHAAGS

116 118 120

116 118 120

116 118 120

A)

Subclass B1 CcrA¹²

Subclass B2 CphA¹³

Subclass B3 L1¹⁴

B)

Fig. 1. A) Comparison of alignment of the amino acid sequences between subclasses

of MBLs

. B) Comparison of the active site of the three subclasses

.

MBL

subclass B1

His116-x-His118-x-Asp120

BcII

1

Zn(II)

2

Zn(II)

Subclass B2

Zn(II)イ

1

2

Zn(II)

2

Zn(II)

Subclass

subclass B1

Zn(II)イオンが Zn2

Zn1

Zn(II)

subclass B3

His121

Zn2

イトの

Zn(II)イオンに配位していることが X

た

(Fig. 1B)

MBL

IMP family

VIM family

P.

aeruginosa

S. marcescens

IMP

VIM

(Fig. 2)

ット遺伝子発現のための

promotor

IMP

VIM

IMP family

IMP-1

IMP-13

VIM-2

VIM-8

IMP family

および

VIM family

MBL

への拡散は

IMP family

VIM family

MBL

MBL

インサーション配列 (IS) インサーション配列 (IS) インテグラーゼ遺伝子

メタロ-β-ラクタマーゼ遺伝子

アミノグリコシド耐性遺伝子

耐性遺伝子カセット

インテグロン構造

伝達性R-プラスミド

耐性菌

インサーション配列 (IS) インサーション配列 (IS)

インテグラーゼ遺伝子

メタロ-β-ラクタマーゼ遺伝子

アミノグリコシド耐性遺伝子

耐性遺伝子カセット

インテグロン構造

伝達性R-プラスミド

耐性菌

Fig. 2. The structure of integron mediated the gene encoded IMP family.

CcrA

3

Fig. 3

MBL

β-

Zn(II)

1

2

X

MBL

β-

Zn(II)

Fig. 3B

1

Zn(II)

3

His

Zn(II)

(Zn2)

1

Asp

Cys

His

染色体

Fig. 3. A) The overall structure of CcrA (PDB code, 1ZNB

). B) The active site of CcrA

MBL

Zn

BcII

Zn

CcrA

BcII

OH

OH

活性中心にある

Asp

β-ラクタム環が加水分解される

19。一方

CcrA

2

Zn(II)イオンに架橋した OH

OH

BcII

Zn2

Asp

フラップ

O O Asp His

Zn1 O H

H

+H₂O

His His

H

S

COO^-

NO₂ O₂N

N S C

O O

Asp His

H

Zn1 His

N S

O COO^- HN

S C

NO₂

NO₂ His

OH

O H

O O

Asp H His

Zn1 His

N S

O COO^- C N

S

NO₂

NO₂ H

O

His

nitrocefin (391 nm) nitrocefin tetrahedral-intermediate

hydrolyzed nitrocefin (491 nm)

O

O H

Fig. 4. Proposed mechanism of nitrocefin hydrolysis by the mono-Zn(II) MBL (BcII).

MBL

1996

trifluoromethyl alcohols and ketones

hydroxamates

thiols

mercaptocarboxylates

1β-methylcarbapenem

2,3-disubstituted succinic acid

tricyclic natural products

N-arylsulfonyl dydrazones

synthetic

cephamycin

類の

MBL

-

MBL

1) Zn(II)イオンおよび近隣のアミノ酸残基、 2)

X

Zn1 Zn2 His Zn2

Zn1 Zn2 His

His

Zn1 His

N S

O

COO^-

NO₂ NO₂ C N

S

H

O

Cys H OH

O

-O Asp

O

H

Cys His

His

-O O Asp His

H

S

COO^-

NO₂ O₂N

N

S C H

O

O H

His

His ^-O O

Asp N O

OH

S

COO^-

NO₂ O2N

N

S C H

O

Cys

His O

H

H

+ H₂O

nitrocefin anion-intermediate (665 nm)

Fig. 5. Proposed mechanism of nitrocefin hydrolysis by the di-Zn(II) MBL (CcrA)

詳細な構造を明らかにした上で設計することが重要である。

MBL-

X

CcrA

4-morpholinoethanesulfonic acid (MES)の複合体

tricyclic natural products

IMP-1

mercaptocarboxylate (2-[5-(1-tetrazolyl-methyl)thien-3-yl]

-N-[2-(mercaptomethyl)-4-(phenylbutyrylglycine)])

succinic acid

Chryseobacterium meningosepticum

BlaB

D-captopril

FEZ-1

D-captopril

X

感染菌の

MBL

重要である。現在知られている

MBL

PCR

Arakawa、Goto

ディスク

(SMA)

(CAZ)

告した⁷⁵。これは、NCCLS法⁷⁶に従いミューラーヒントン培地に

MacFarland 0.5

MBL

SMA

CAZ

MBL

これらの背景の下、本研究は日本で発見され、その耐性遺伝子がプラスミド上に存

在する

IMP-1

2

IMP-1

Zn(II)

イオンの解離機構をイミペネム加水分解活性の

pH

3

MBL

Asp120

IMP-1

pH

速度論的解析および

X

Asp120

第

4

IMP-1

IMP-1

害剤を設計、合成し阻害活性ならびに阻害様式を速度論的解析、

MALDI-TOF MS

X

5

ーブ法で

IMP-1

定および阻害活性測定から新規蛍光剤の特徴づけを行なった。以下に得られた知見を 詳述する。

第

2

metallo-β-lactamase (IMP-1)からの Zn(II)イオンの解離

β-

pH

第

1

加水分解酵素の研究において、活性の

pH

1

pK

金属イオンの解離がリガンドのプロトン化状態に依存するため、

pH

(

)

OH

H

OH

pK

加水分解酵素にとって、如何に攻撃種である

OH

pK

OH

pK

1

Zn(II)イオンへの配位がある。

これが多くの加水分解酵素の活性中心に

Zn(II)

1

OH

pK

14.0

pK

10.0

Zn(II)イオ

OH

H

Zn(II)-OH

Zn(II)-OH

pK

carboxypeptidase A

Zn(II)-OH

の

pK

6.1

本研究対象である

MBL

Zn

β-

2

Zn(II)イオンに架

橋して配位している配位水(Zn1-OH2

-Zn2)だと考えられている

Zn

BcII

β-

2

H

1

Zn(II)イオンに配位している Zn1-OH

pK

5.6

1

H

MBL

Asp

pK

MBL

Zn

CcrA

pH

pK

5

9

2

Zn(II)イオンに架橋した Zn1-OH

-Zn2

pK

5.0

22, 23。これらのことから単核

Zn

BcII

Zn

CcrA

が提案された⁵³。

しかし

BcII

CcrA

pH

Zn(II)

Zn(II)イ

(2)

OH

(Zn-OH

)

ン化され

OH

(Zn-OH

)になると活性が低下すること、以上 2

当な評価ができていないと考えた。

そこで、MBLであり複核

Zn

IMP-1

pH

1

pH

IMP-1

Zn(II)

2）(1)を基にしたイミペネム加水分解活性の pH

を行なった。この結果を基に、

IMP-1

Zn

Fig. 6. Time-course of absorbance change at 298 nm at 30 ºC for hydrolysis of imipenem on the addition of 10 nM IMP-1 in 50 mM Tris buffer(pH 7.4) to 100 µM imipenem in 0.2 M acetic acid-sodium acetate buffer(pH 5.1)-0.5 M NaCl. After 900 s, Zn(NO

)

was added and the measurement was continued.

第

2

2-1 酸性溶液中における IMP-1

Zn(II)イオン添加による

活性の回復

酸性溶液中における

IMP-1

100 µM

IMP-1

7.4, 0.5 M NaCl)

10 nM

298 nm

100

100

反応が終了したかのように見えた(Fig. 6)。

Fig. 7. Dependence of activity, k

, for hydrolysis of imipenem by IMP-1 on the concentration of Zn(NO

)

at pH 5.0.

しかし今回用いたイミペネムがすべて分解された場合、その吸光度変化は

1.0

400

0.1

900

Zn(NO

)

100 µM

298 nm

(Fig. 6)

2-2

IMP-1

Zn(II)

2-1

IMP-1

Zn(II)

失することがわかった。このことから活性のある

Zn

Zn

Zn(II)

IMP-1

pH 4.0

9.0

30 ºC

10

Zn(II)

v

Table 1. The dissociation constants of Zn(II) from IMP-1 and the limiting k

for the hydrolysis with concentration of Zn(II) determined using imipenem as a reporter substrate.

pH k

/min

K

/µM log k

p(K

/µM)

4.0 200 6100 2.29 -3.79

4.1 550 7000 2.74 -3.85

4.2 770 2900 2.89 -3.46

4.33 990 840 2.99 -2.93

4.45 1210 180 3.08 -2.26

4.55 1370 84 3.14 -1.92

4.73 1450 16 3.16 -1.21

5.0 1670 2.6 3.22 -0.42

5.21 1790 1.3 3.25 -0.10

5.41 1910 0.38 3.28 0.42

5.61 2660 0.70 3.43 0.16

5.61 3380 0.11 3.53 0.97

6.0 3900 0.19 3.59 0.73

7.4 5210 0.25 3.72 0.60

8.0 5160 0.093 3.71 1.03

9.0 4950 0.17 3.69 0.78

から酵素濃度[E]Tで除することで分子活性

k

(v

/[E]

)を得た。 Fig. 7

pH 5.0

る

Zn(II)

k

Zn(II)

の

v

82 min

k

Zn(II)イオン濃度の増加により上

Zn(II)

0.5 µM

320 min

20 µM

= 1500 min

) (Fig. 7)。

この挙動は酸性溶液中においては、

Zn(II)

Zn

Zn

IMP-1

Zn(II)

K

k

は、Eq. 9 (p74参照)の非線形最小二乗法を使って求めた。得られた速度論的パラメー タ

K

k

Table 1

K

pH 5.0

4.0

K

K

pH

(Fig. 8)

Fig. 8

pH

-log K

pH

Fig. 8. Dependence of dissociation constant (K

) (○) and limiting rate constant (k

) (□) for imipenem by IMP-1 on pH.

5.0

4.0

Fig. 8

Zn(II)

pK

5.3

2-3 イミペネムに対する加水分解の pH

活性変化から得られた

IMP-1

Zn(II)イオンの解離定数 K

2-2

IMP-1

pH

pH

K

25

Zn(NO

)

の濃度で基質であるイミペネムの濃度を変えて分子活性

k

pH 4.0

5.6

(0.2 M, 0.5 M NaCl)

pH 5.6

9.0

MTEN

Michaelis-Menten

K

k

Fig. 9

log k

pH

pH 7.0

8.0

Fig. 9. pH profiles of log (k

/min

) (○), log (K

/µM) (□) and log (k

/K

/min

µM) (∆) for the hydrolysis of imipenem by IMP-1 under sufficient concentration of Zn(II) at 30 ºC.

の間で最大となったが、pH 4.0と

5.0

K

6.0

pH 4

log K

/k

8.0

第

3

加水分解酵素の活性の

pH

MBL

β-

剤の加水分解の

pH

pH

Zn(II)

10

500

pH

2

BcII

2

Zn(II)

Zn1-OH

Asp

pH

Zn(II)イオンに配位し

pH 7.4

IMP-1

pH 5.1

始まると基質に対する活性は

100

900

Zn(II)イオンを添加したと

Zn(II)

IMP-1

pH 7.4

pH 5.0

ある速い変化が起こったため、数

100

100

Eq. 1

active form non-active form + Zn(II) Eq. 1 K

酸性領域において、活性から求められた

Zn(II)イオンの解離定数 K

pH

4

Zn(II)

pK

5.3

Zn(II)イオンリガンドのプロトン化は、酸性溶液中においてみか

けの

IMP-1

Zn(II)

位は

pH

をしている。遊離の

His

pK

5.97

His

Nδ

pK

pH

Zn(II)

IMP-1

Zn

1

2

IMP-1

Zn

MBL

Zn(II)イオ

IMP-1

Zn(II)

Eqs. 2, 3

E-Zn

E-Zn + Zn

E-Zn E + Zn

K

K

Eq. 2 Eq. 3

不活性なフリーの酵素濃度[E]、mono-Zn(II)型酵素濃度[E-Zn]と

di-Zn(II)型酵素濃度 [E-Zn

]

Eqs. 4-6

[E] = [E]

1 + K

[Zn] +

K

K

[Zn]

[E-Zn] = [E]

[Zn]/K

1 + K

[Zn] +

K

K

[Zn]

[E-Zn

] = [E]

[Zn]

/K

K

1 + K

[Zn] +

K

K

[Zn]

Eq. 4

Eq. 5

Eq. 6

非線形最小二乗法を用いて

Eq. 9

di-Zn(II)

IMP-1(E-Zn

)

2

Zn(II)

て、フリーの

IMP-1(E)になったため、 IMP-1

E-Zn2

E-Zn

E

IMP-1

2

Zn(II)イオンが協同的に IMP-1

た。それゆえに、本研究で得られた

K

Eq. 3

K

また酸性領域で、

pH

4.0

Zn(II)

2

Zn(II)イオンに配位している Zn1-OH

-Zn2、

pK

5.97

His

Nδ

pK

3.96

Asp

Oδ1

Oδ2

4

His

1

Asp

Zn(II)

Asp

Oδ1

Oδ2

pK

が

3.96

5.3

IMP-1

Zn(II)イオンは、 His

Nδ

pK

pH

過剰の

Zn(II)

log k

Fig. 9

pH

Good buffer

MOPS

MES

Fitzgerald

CcrA

IMP-1

k

5

0.6

2

Zn(II)

Zn1-OH

-Zn2

pK

5

pK

MBL

1

BcII

CcrA

Zn(II)

1

2

Zn1-OH

Zn1-OH

-Zn2

human carbonic

anhydrase II

Zn(II)

Zn-OH

pK

6.8

Zn-OH

pK

10

CcrA

今回の研究と同様に

pH 5

9

pK

Zn(II)

OH

pK

2

Zn(II)

Zn1-OH

-Zn2

mono-Zn(II)酵素より di-Zn(II)酵素

第

4

本章では、

MBL

IMP-1

IMP-1

Zn(II)

pH

pH

(1) IMP-1

Zn(II)

とがわかった。

(2) IMP-1

Zn(II)イオンの解離は pH

4

pK

5.3

(3) IMP-1

pH

pH 5

9

pK

β-ラクタム環のカルボニル炭素を求核攻撃すると考えられている Zn1-OH

-Zn2

pK

5

以上の結果から、

IMP-1

β-

Zn1-OH

-Zn2

pK

5

Zn1-OH

-Zn2

CcrA

IMP-1