suppmat1559.doc 198KB Jun 05 2011 09:30:54 PM
SUPPLEMENTARY MATERIAL
Table 4
Multiple Amino-Acid Sequence Alignment for 45 Class A -Lactamases (Positions 214 and 233
are marked by asterisks)
Amino Acid Position
200
210
220
230
..|....... ..|...*... ..|.....-- ....|..*..
B. fragilis CS30
--DKEYKNFI YQTMVECQTG QDRLIAPLLD K-KVTMGHKT
B. uniformis WAL–7088
--NKELKDFL WQTMIDTETG ANKLKG-MLP A-KTVVGHKT
PER-1, P. aeruginosa RNL-1
--ETS-QALL WKWMVETTTG PERLKG-LLP A-GTVVAHKT
B. vulgatus CLA341
--DDEKQSFI KNTLKECKTG VDRIAAPLLD KEGVVIAHKT
OHIO-1, E. cloacae
RLSARSQRQL LQWMVDDRVA GRLIRSVL-- PAGWFIADKT
SHV, K. pneumoniae
RLSARSQRQL LQWMVDDRVA GPLIRSVL-- PAGWFIADKT
LEN-1, K. pneumoniae LEN-1
HLSARSQQQL LQWMVDDRVA GPLIRAVL-- PPGWFIADKT
TEM, E. coli
LLTLASRQQL IDWMEADKVA GPLLRSAL-- PAGWFIADKS
P. mirabilis GN179
TLSIKSRQQL ESWLKGNEVG DALFRKGV-- PSDWIVADRT
PSE, P. aeruginosa
ALSEMNQKKL ESWMVNNQVT GNLLRSVL-- PAGWNIADRS
R. capsulatus SP108
VLSPEARGKL AEWMRHGGVT GALLRAEA-- EDAWLILDKS
NMC, E. cloacae NOR-1
ILSEHEKETY QTWLKGNTTG AARIRASV-- PSDWVVGDKT
Sme-1, S. marcescens S6
VLNAKVKAIY QNWLKGNTTG DARIRASV-- PADWVVGDKT
OXY-2, K. oxytoca D488
ALGEQQRAQL VTWLKGNTTG GQSIRAGL-- PESWVVGDKT
K. oxytoca E23004/SL781/SL7811 ALGEQQRAQL VTWLKGNTTG GQSIRAGL-- PASWAVGDKT
S. typhimurium CAS-5
ALAETQRAQL VTWLKGNTTG SASIRAGL-- PKSWVVGDKT
MEN-1, E. coli MEN
ALGDSQRAQL VTWMKGNTTG AASIQAGL-- PASWVVGDKT
S. fonticola CUV
ALAEPQRAQL VEWMKGNTTG GASIRAGL-- PTTWVVGDKT
C. diversus ULA27
ALAGAQRAQL VEWLKGNTTG GQSIRAGL-- PEGWVVGDKT
Proteus vulgaris 5E78-1
ALGQSQRQQL VTWLKGNTTG DHSIKAGL-- PKHWIVGDKT
B. cepacia 249
ALPPAQRAQL IEWLRGNKVG DKRIRAGV-- PTGWRVGDKT
Y. enterocolitica serotype 0:3/Y-56 ALRPAQRSQL AVWLKGNTTG DATIRAGA-- PTDWIVGDKT
M. tuberculosis H37RV
ALPPDKRALL TDWMARNTTG AKRIRAGF-- PADWKVIDKT
S. clavuligerus NRRL 3585
ALTPDRRALL TDWLVRNTTG GRRIRAGV-- PSGWRVADKT
III, B. cereus 569/H
VLPSEKRELL VDWMKRNTTG DKLIRAGV-- PKGWEVADKT
B. licheniformis 749/C
KLPSEKRELL IDWMKRNTTG DALIRAGV-- PDGWEVADKT
I, B. mycoides NI10R
ALPAEKRKIL TEWMKGNATG DKLIRAGV-- PTDWIVGDKS
I, B. cereus 569/H/9
ALPAEKRKIL TEWMKGNATG DKLIRAGI-- PTDWVVGDKS
I, B. cereus 5/B
ALPHQKRNIL TEWMKGNATG DKLIRAGV-- PTDWVDADKS
B. subtilis 168/6GM
TLTTDKRMIL TDWMRGNATG DELIRAGA-- PIGWEVGDKS
2, S. cacaoi DSM40057
VLAEGDRKQL TTWLRNNTTG DGLIRAGV-- RQGWVVGDKT
S. badius DSM40139
ALRAPERAQL TTWLRTNTTG DAVIRAGV-- PENWVVGDKT
Actinomadura sp. R39
VLEEGPRDVL TEMLLNNTTG DELIRAGV-- PEDWRVGDKT
N. lactamdurans LC411
ALAEDDRALL TDWLRRNTTG GTVIRAGV-- PADWVVGDKT
S. cacaoi KCC S0352
ALAPNDREQL NDWMSGSRTG DALIRAGV-- PKDWKVEDKS
ROB-1, H. influenzae F990
TLTESQKTIL WNWLDNNATG NPLIRAAT-- PTSWKVYDKS
S. fradiae DSM40063
LLAAHDRERL TRWMLDNRTS DERFRKGL-- PADWLLADKT
S. lavendulae DSM2014
ALAPRDRERL TGWLLANTTS TERFRKGL-- PADWTLGDKT
S. albus G
ALNPRDRRLL TSWLLANTTS GDRFRAGL-- PDDWTLGDKT
S. lavendulae KCCS0263
GLGRADRGLL TGWLCANTTS GARFRKGL-- PAEWRLGDKT
Source
S. aureofaciens
S. cellulosae KCCS0127
M. fortuitum
S. aureus PC1/SK456/NCTC9789
BRO-1, M. catarrhalis ATCC53879
ALRAGDRKRL TGWLVANTTN RPTFRAGL-- PDDWVLADKT
ALNRPDRALL TDWLLRNTTT LTTFRTGL-- PKGWTVADKS
ALSPPQRGLL EDWMRANQTS S--MRAGL-- PEGWTTADKT
-LSKENKKFL LDLMLNNKSG DTLIKDGV-- PKDYKVADKS
ILSNQSKEQL ITWLINDKVA DNLLRKYL-- PKNWRIGDKT
Table 5
Calculated pKa and pKint for Some Titratable Residues from the Active Site of E. coli TEM-1 (1btl).
Parameter Set
His
εin
tautomerism
pKa(pKint)
pI
Ser-70 (A)
Lys-73 (C)
Glu-166 (A) Lys-234 (C) Arg-244 (C) Asp-214 (A)
Asp-233 (A)
AMBER91
AMBER91
Yes
Yes
20 4.65
4 4.49
23.7 (12.7) 13.5 (7.89) 0.561 (3.39) 9.31 (7.44) 11.7 (9.73)
>30 (13.3) 13.9(.0043) -0.004 (3.98) -5.53 (-2.11) 5.53 (4.25)
1.92 (3.26)
29.8 (5.67)
-2.02 (2.16)
30 (10.3)
2.64 (5.64) 13.5 (10.8) 14.8 (12.3)
2.46 (11.2) 9.78 (10.0) 13.1 (11.5)
5.18 (5.34)
>30 (12.3)
-0.55 (3.72)
-8.50 (4.76)
PARSE3
PARSE3
No
No
20 5.04 23.8 (14.8) 15.7 (10.2)
4 5.34 >21.5 (18.5) >30 (7.40)
2.91 (5.03) 12.9 (10.4) 16.1 (13.1)
6.29 (9.80) 7.51 (8.09) 19.8 (15.8)
3.37 (5.15)
17.2 (12.3)
0.901 (5.49)
-2.61 (14.0)
AMBER91
AMBER91
No
No
20 4.46
4 4.07
23.8 (12.7) 13.5 (7.77) 0.627 (3.27) 9.19 (7.27) 11.6 (9.67)
>30 (13.3) 15.3 (-0.63) -0.528 (3.52) 30 (9.03)
2.77 (5.16) 13.5 (10.5) 14.8 (12.3)
3.85 (9.88) 8.11 (8.48) 13.3 (11.4)
3.79 (5.16)
≈22 (12.0)
0.745 (4.66)
-3.22 (9.90)
AMBER94
AMBER94
No
No
20 5.06
4 5.09
24.9 (15.0) 16.6 (10.4)
21.2 (19.4) >30 (7.99)
2.85 (4.80) 13.3 (10.4) 28.2 (19.4)
2.82 (8.07) 17.3 (8.34) >30 (47.2)
3.32 (5.00)
17.3 (11.3)
0.445 (4.46)
-4.02 (9.01)
E. coli TEM-1 pI is 5.4 (Labia, R.; Lelievre, V.; Peduzzi, J. Biochim. Biophys. Acta 1980, 611, 351-357)
Table 6
Calculated pKa and pKint for Some Titratable Residues from the Active Site of B. licheniformis 749/C Wild Type (4blm) and Glu166Ala
Mutant (1mbl) Compared with E. coli TEM-1 (1btl).
Enzyme
εin
pKa (pKint)
pI
Ser-70 (A)
Lys-73 (C) Glu-166 (A) Lys-234 (C) Arg-244 (C) Asp-214 (A)
Asp-233 (A)
BCL w.t.
BCL w.t.
20
4
4.56
4.99
25.2 (14.9)
26.1 (19.3)
17.1 (10.6) 2.28 (4.64)
>30 (9.56) 1.43 (7.73
14.2 (10.6)
13.6 (9.09)
17.4 (11.9) Asn-214
20.4 (9.76) Asn-214
-0.771 (3.99)
-8.39 (6.63)
BCL E166A
BCL E166A
20
4
4.54
4.87
20.0 (14.2)
10.0 (16.2)
12.6 (10.2)
28.6 (8.49)
14.1 (10.4)
18.7 (8.34)
17.1 (11.7) Asn-214
18.9 (9.36) Asn-214
-0.972 (3.96)
-8.96 (6.80)
TEM-1
TEM-1
20
4
5.06
5.09
24.9 (15.0)
21.2 (19.4)
16.6 (10.4) 2.85 (4.80)
>30 (7.99) 2.82 (8.07)
13.3 (10.4)
17.3 (8.34)
28.2 (19.4)
>30 (47.2)
Ala-166
Ala-166
3.32 (5.00)
17.3 (11.3)
0.445 (4.46)
-4.02 (9.01)
B. licheniformis 749/C pI is 5.13 (Imanaka, T.; Oshihara, W.; Himeno, T.; Aiba, S. J. Gen. Microbiol., 1983, 129, 2621-2628.)
Table 7
Calculated pKa and pKint for Some Titratable Residues from the Active Site of S. aureus PC1 Wild Type (3blm) and Lys73His Mutant
(1dja) Compared with E. coli TEM-1 (1btl).
pKa (pKint)
Enzyme
εin
pI
SA PC1
SA PC1
20
4
10.0
9.52
SA PC1 K73H 20
SA PC1 K73H 4
ND
ND
24.3 (14.8) 17.9 (10.7)
1.01 (4.50)
21.5 (18.9) >30 (9.96)
-1.32 (6.92)
His-73 (H; H )
25.3 (16.3) 6.87 (6.03; 6.05) 0.614 (6.14)
>30 (23.4) 8.01 (2.08; 3.47) -3.34 (13.2)
5.06
5.09
Lys-73(C)
24.9 (15.0) 16.6 (10.4)
21.2 (19.4) >30 (7.99)
TEM-1
TEM-1
20
4
Ser-70 (A)
Lys-73(C)
Glu-166 (A) Lys-234 (C) Arg-244 (C) Asp-214 (A) Asp-233 (A)
2.85 (4.80)
2.82 (8.07)
14.2 (10.5)
13.6 (916)
>30 (19.1)
>30 (45.3)
Asn-214
Asn-214
0.027 (4.66)
-3.22 (9.62)
17.2 (12.4)
26.4 (15.9)
17.4 (12.5)
16.9 (10.5)
Asn-214
Asn-214
1.07 (5.23)
~-1 (11.4)
13.3 (10.4)
17.3 (8.34)
28.2 (19.4)
>30 (47.2)
3.32 (5.00) 0.445 (4.46)
17.3 (11.3) -4.02 (9.01)
S. aureus PC1 pI is 10.1 (Zygmunt, D. J.; Stratton, C. W.; Kernodle, D. S. Antimicrob. Agents. Chemother. 1992, 36, 440-445.)
Table 8
Calculated pKa and pKint for Some Titratable Residues from the Active Site of the Model of the Michaelis Complex of E. coli TEM-1,
B. licheniformis 749/C Wild Type, its Glu166Ala Mutant, S. aureus PC1 Wild Type and its Lys73His Mutant with Penicillin G.
pKa (pKint)
Enzyme
εin Ser-70 (A)
Lys-73(C)
Glu-166 (A)
Lys-234 (C) Arg-244 (C) Asp-214 (A) Asp-233 (A)
12.5 (6.91) 13.9 (9.15) 2.05 (3.07) -1.57 (2.57)
3.80 (-6.19) 10.7 (-.35) >17 (5.12) -9.95 (2.03)
TEM-1 (91)
TEM-1 (91)
20 27.9 (12.4) 16.1 (7.02)
4 >28 (13.2) >30 (-5.56)
-0.662 (2.72)
-8.21 (1.96)
TEM-1
TEM-1
20 29.5 (15.4) 17.9 (9.96)
4 >30 (25.0) >30 (6.35)
0.785 (4.15)
-1.72 (6.88)
18.5 (10.9)
>30 (10.2)
23.5 (16.1) 4.93 (5.40) 0.213 (4.59)
>30 (29.4) 16.3 (13.2) -4.34 (9.54)
BCL w.t.
BCL w.t.
20 >30 (15.5) 19.4 (10.6)
4 >30 (25.0) >30 (9.19)
0.669 (4.07)
-2.95 (6.23)
19.5 (10.7)
>30 (9.34)
19.6 (11.7) Asn-214
26.6 (8.60) Asn-214
3.82 (3.48)
30 (8.74)
19.2 (11.8) Asn-214
26.6 (8.78) Asn-214
-2.52 (3.38)
30 (15.4) 19.8 (10.1)
4 >30 (24.4) >30 (6.70)
His -73 (H; H)
20 >30 (16.7) 5.56 (4.39; 5.02)
4 >30 (29.2) 30 (14.2) -5.64 (0.607) 23.0 (12.1) 6.68 (3.15)
Intact
Yes
20 8.45 CHARMM22
26.0 (14.5) 15.4 (9.70) 7.76 (12.1) 21.7 (10.7)
Intact
Yes
4 7.77 CHARMM22
>20 (17.1) -1.49 (5.18) >18 (18.4) >14 (10.2)
Intact
No
20 6.46 AMBER91
26.5 (12.3) 14.3 (7.24) 5.23 (9.01) 12.2 (7.18)
Intact
No
4 4.88 AMBER91
>30 (12.8) -5.93 (-1.02) 21.2 (10.3) 4.17 (.811)
Intact
No
20 8.49 AMBER94
25.8 (14.4) 15.0 (9.40) 8.12 (11.5) 19.6 (10.5)
Intact
No
4 7.60 AMBER94
>30 (16.9) -3.52 (3.96) 12.9 (16.2) >30 (9.43)
Intact
No
20 8.45 CHARMM22
26.5 (14.5) 15.1 (9.51) 7.76 (11.6) 20.3 (10.5)
Intact
No
4 7.69 CHARMM22 ≈26.3 (17.1) -2.76 (4.32) 14.7 (16.7) >30 (9.57)
Intact
No
20 8.63
PARSE3
23.1 (14.0) 12.4 (9.66) 8.36 (11.4) 19.8 (10.7)
Intact
No
4 7.64
PARSE3
23.5 (15.5) -1.01 (5.51) 13.6 (16.1) >30 (10.6)
Michaelis
No
20 ND
AMBER91
>30 (13.8) 18.8 (8.68) 10.4 (9.52) 25.0 (7.56)
Michaelis
No
4 ND
AMBER91
>30 (19.7) 4.97 (2.17) >30 (11.0)
Michaelis
No
20 ND
AMBER94
>30 (16.2) 20.6 (10.7) 12.2 (12.0) 26.8 (10.6)
Michaelis
No
4 ND
AMBER94
>30 (28.4) 12.2 (9.95) 19.1 (19.5) >30 (9.71)
≈30 (-1.38)
E. cloacae P99 pI = 8.2; 7.8; 8.3; 8.25; 8.95 (Goering, R. V.; Sanders, C. C.; Sanders, W. E., Jr.;
Guay, R.; Guerin, S. Rev. Infect. Dis. 1988, 10, 786-792; Goward, C. R.; Stevens, G. B.;
Hammond, P. M.; Scawen, M. D. Chromatogr. 1988, 457, 317-324; Minami, S.; Inoue, M.;
Mitsuhashi, S. Antimicrob. Agents. Chemother. 1980, 18, 853-857; Seeberg, A. H.; TolxdorffNeutzling, R. M.; Wiedemann, B. Antimicrob. Agents. Chemother. 1983, 23, 918-925.)
Calculated sequence-based pI is 8.24 for positions 22-380 as in x-ray sequence.
Table 10
Calculated pKa and pKint for Some Titratable Residues from the Active Site of the DDTranspeptidase R61 Streptomyces sp. (1cef) and a Model of its Michaelis Complex with
Penicillin G.
Enzyme His εin pI
tautomerism
Parameter
Set
pKa (pKint)
Ser-62 (A)
Lys-65 (C)
Tyr-159 (A)
18.1 (8.38)
7.69 (10.2)
-1.37 (3.79)
27.0
9.21
7.84
9.31
>24
9.23
7.48
9.20
6.92
30
>30
>30
28.3
18.5
≈30
27.9
>30
29.8
(14.0) -1.97 (1.71)
(14.5) 23.6 (10.3)
(17.0) 13.1 (7.98)
(14.1) 21.9 (10.3)
(14.7) 8.55 (8.45)
(14.3) 21.8 (10.1)
(16.2) 12.9 (7.27)
(16.4) 20.5 (12.6)
(21.3) ~10.5 (15.7)
Michaelis
Michaelis
Michaelis
Michaelis
Michaelis
Michaelis
No
No
No
No
Yes
Yes
20
4
20
4
20
4
ND
ND
ND
ND
ND
ND
>30
>30
>30
>30
>30
>30
(13.2)
(16.1)
(14.8)
(21.0)
(17.8)
(33.1)
AMBER91
AMBER91
AMBER94
AMBER94
AMBER94
AMBER94
24.7
5.81
25.8
20.7
25.9
>18
(13.0)
(11.6)
(17.3)
(11.4)
(16.5)
(11.2)
(15.1)
(13.7)
(22.6)
His-298 (C)
(-3.44)
(6.39)
(6.92)
(6.24)
(5.43)
(5.78)
(4.25)
(7.94;7.26)
(9.72; 8.22)
(8.30) 14.1 (9.82) -3.58 (1.36)
(-0.21) >30 (12.6) 30 (17.3) 30 (28.4)
Table 4
Multiple Amino-Acid Sequence Alignment for 45 Class A -Lactamases (Positions 214 and 233
are marked by asterisks)
Amino Acid Position
200
210
220
230
..|....... ..|...*... ..|.....-- ....|..*..
B. fragilis CS30
--DKEYKNFI YQTMVECQTG QDRLIAPLLD K-KVTMGHKT
B. uniformis WAL–7088
--NKELKDFL WQTMIDTETG ANKLKG-MLP A-KTVVGHKT
PER-1, P. aeruginosa RNL-1
--ETS-QALL WKWMVETTTG PERLKG-LLP A-GTVVAHKT
B. vulgatus CLA341
--DDEKQSFI KNTLKECKTG VDRIAAPLLD KEGVVIAHKT
OHIO-1, E. cloacae
RLSARSQRQL LQWMVDDRVA GRLIRSVL-- PAGWFIADKT
SHV, K. pneumoniae
RLSARSQRQL LQWMVDDRVA GPLIRSVL-- PAGWFIADKT
LEN-1, K. pneumoniae LEN-1
HLSARSQQQL LQWMVDDRVA GPLIRAVL-- PPGWFIADKT
TEM, E. coli
LLTLASRQQL IDWMEADKVA GPLLRSAL-- PAGWFIADKS
P. mirabilis GN179
TLSIKSRQQL ESWLKGNEVG DALFRKGV-- PSDWIVADRT
PSE, P. aeruginosa
ALSEMNQKKL ESWMVNNQVT GNLLRSVL-- PAGWNIADRS
R. capsulatus SP108
VLSPEARGKL AEWMRHGGVT GALLRAEA-- EDAWLILDKS
NMC, E. cloacae NOR-1
ILSEHEKETY QTWLKGNTTG AARIRASV-- PSDWVVGDKT
Sme-1, S. marcescens S6
VLNAKVKAIY QNWLKGNTTG DARIRASV-- PADWVVGDKT
OXY-2, K. oxytoca D488
ALGEQQRAQL VTWLKGNTTG GQSIRAGL-- PESWVVGDKT
K. oxytoca E23004/SL781/SL7811 ALGEQQRAQL VTWLKGNTTG GQSIRAGL-- PASWAVGDKT
S. typhimurium CAS-5
ALAETQRAQL VTWLKGNTTG SASIRAGL-- PKSWVVGDKT
MEN-1, E. coli MEN
ALGDSQRAQL VTWMKGNTTG AASIQAGL-- PASWVVGDKT
S. fonticola CUV
ALAEPQRAQL VEWMKGNTTG GASIRAGL-- PTTWVVGDKT
C. diversus ULA27
ALAGAQRAQL VEWLKGNTTG GQSIRAGL-- PEGWVVGDKT
Proteus vulgaris 5E78-1
ALGQSQRQQL VTWLKGNTTG DHSIKAGL-- PKHWIVGDKT
B. cepacia 249
ALPPAQRAQL IEWLRGNKVG DKRIRAGV-- PTGWRVGDKT
Y. enterocolitica serotype 0:3/Y-56 ALRPAQRSQL AVWLKGNTTG DATIRAGA-- PTDWIVGDKT
M. tuberculosis H37RV
ALPPDKRALL TDWMARNTTG AKRIRAGF-- PADWKVIDKT
S. clavuligerus NRRL 3585
ALTPDRRALL TDWLVRNTTG GRRIRAGV-- PSGWRVADKT
III, B. cereus 569/H
VLPSEKRELL VDWMKRNTTG DKLIRAGV-- PKGWEVADKT
B. licheniformis 749/C
KLPSEKRELL IDWMKRNTTG DALIRAGV-- PDGWEVADKT
I, B. mycoides NI10R
ALPAEKRKIL TEWMKGNATG DKLIRAGV-- PTDWIVGDKS
I, B. cereus 569/H/9
ALPAEKRKIL TEWMKGNATG DKLIRAGI-- PTDWVVGDKS
I, B. cereus 5/B
ALPHQKRNIL TEWMKGNATG DKLIRAGV-- PTDWVDADKS
B. subtilis 168/6GM
TLTTDKRMIL TDWMRGNATG DELIRAGA-- PIGWEVGDKS
2, S. cacaoi DSM40057
VLAEGDRKQL TTWLRNNTTG DGLIRAGV-- RQGWVVGDKT
S. badius DSM40139
ALRAPERAQL TTWLRTNTTG DAVIRAGV-- PENWVVGDKT
Actinomadura sp. R39
VLEEGPRDVL TEMLLNNTTG DELIRAGV-- PEDWRVGDKT
N. lactamdurans LC411
ALAEDDRALL TDWLRRNTTG GTVIRAGV-- PADWVVGDKT
S. cacaoi KCC S0352
ALAPNDREQL NDWMSGSRTG DALIRAGV-- PKDWKVEDKS
ROB-1, H. influenzae F990
TLTESQKTIL WNWLDNNATG NPLIRAAT-- PTSWKVYDKS
S. fradiae DSM40063
LLAAHDRERL TRWMLDNRTS DERFRKGL-- PADWLLADKT
S. lavendulae DSM2014
ALAPRDRERL TGWLLANTTS TERFRKGL-- PADWTLGDKT
S. albus G
ALNPRDRRLL TSWLLANTTS GDRFRAGL-- PDDWTLGDKT
S. lavendulae KCCS0263
GLGRADRGLL TGWLCANTTS GARFRKGL-- PAEWRLGDKT
Source
S. aureofaciens
S. cellulosae KCCS0127
M. fortuitum
S. aureus PC1/SK456/NCTC9789
BRO-1, M. catarrhalis ATCC53879
ALRAGDRKRL TGWLVANTTN RPTFRAGL-- PDDWVLADKT
ALNRPDRALL TDWLLRNTTT LTTFRTGL-- PKGWTVADKS
ALSPPQRGLL EDWMRANQTS S--MRAGL-- PEGWTTADKT
-LSKENKKFL LDLMLNNKSG DTLIKDGV-- PKDYKVADKS
ILSNQSKEQL ITWLINDKVA DNLLRKYL-- PKNWRIGDKT
Table 5
Calculated pKa and pKint for Some Titratable Residues from the Active Site of E. coli TEM-1 (1btl).
Parameter Set
His
εin
tautomerism
pKa(pKint)
pI
Ser-70 (A)
Lys-73 (C)
Glu-166 (A) Lys-234 (C) Arg-244 (C) Asp-214 (A)
Asp-233 (A)
AMBER91
AMBER91
Yes
Yes
20 4.65
4 4.49
23.7 (12.7) 13.5 (7.89) 0.561 (3.39) 9.31 (7.44) 11.7 (9.73)
>30 (13.3) 13.9(.0043) -0.004 (3.98) -5.53 (-2.11) 5.53 (4.25)
1.92 (3.26)
29.8 (5.67)
-2.02 (2.16)
30 (10.3)
2.64 (5.64) 13.5 (10.8) 14.8 (12.3)
2.46 (11.2) 9.78 (10.0) 13.1 (11.5)
5.18 (5.34)
>30 (12.3)
-0.55 (3.72)
-8.50 (4.76)
PARSE3
PARSE3
No
No
20 5.04 23.8 (14.8) 15.7 (10.2)
4 5.34 >21.5 (18.5) >30 (7.40)
2.91 (5.03) 12.9 (10.4) 16.1 (13.1)
6.29 (9.80) 7.51 (8.09) 19.8 (15.8)
3.37 (5.15)
17.2 (12.3)
0.901 (5.49)
-2.61 (14.0)
AMBER91
AMBER91
No
No
20 4.46
4 4.07
23.8 (12.7) 13.5 (7.77) 0.627 (3.27) 9.19 (7.27) 11.6 (9.67)
>30 (13.3) 15.3 (-0.63) -0.528 (3.52) 30 (9.03)
2.77 (5.16) 13.5 (10.5) 14.8 (12.3)
3.85 (9.88) 8.11 (8.48) 13.3 (11.4)
3.79 (5.16)
≈22 (12.0)
0.745 (4.66)
-3.22 (9.90)
AMBER94
AMBER94
No
No
20 5.06
4 5.09
24.9 (15.0) 16.6 (10.4)
21.2 (19.4) >30 (7.99)
2.85 (4.80) 13.3 (10.4) 28.2 (19.4)
2.82 (8.07) 17.3 (8.34) >30 (47.2)
3.32 (5.00)
17.3 (11.3)
0.445 (4.46)
-4.02 (9.01)
E. coli TEM-1 pI is 5.4 (Labia, R.; Lelievre, V.; Peduzzi, J. Biochim. Biophys. Acta 1980, 611, 351-357)
Table 6
Calculated pKa and pKint for Some Titratable Residues from the Active Site of B. licheniformis 749/C Wild Type (4blm) and Glu166Ala
Mutant (1mbl) Compared with E. coli TEM-1 (1btl).
Enzyme
εin
pKa (pKint)
pI
Ser-70 (A)
Lys-73 (C) Glu-166 (A) Lys-234 (C) Arg-244 (C) Asp-214 (A)
Asp-233 (A)
BCL w.t.
BCL w.t.
20
4
4.56
4.99
25.2 (14.9)
26.1 (19.3)
17.1 (10.6) 2.28 (4.64)
>30 (9.56) 1.43 (7.73
14.2 (10.6)
13.6 (9.09)
17.4 (11.9) Asn-214
20.4 (9.76) Asn-214
-0.771 (3.99)
-8.39 (6.63)
BCL E166A
BCL E166A
20
4
4.54
4.87
20.0 (14.2)
10.0 (16.2)
12.6 (10.2)
28.6 (8.49)
14.1 (10.4)
18.7 (8.34)
17.1 (11.7) Asn-214
18.9 (9.36) Asn-214
-0.972 (3.96)
-8.96 (6.80)
TEM-1
TEM-1
20
4
5.06
5.09
24.9 (15.0)
21.2 (19.4)
16.6 (10.4) 2.85 (4.80)
>30 (7.99) 2.82 (8.07)
13.3 (10.4)
17.3 (8.34)
28.2 (19.4)
>30 (47.2)
Ala-166
Ala-166
3.32 (5.00)
17.3 (11.3)
0.445 (4.46)
-4.02 (9.01)
B. licheniformis 749/C pI is 5.13 (Imanaka, T.; Oshihara, W.; Himeno, T.; Aiba, S. J. Gen. Microbiol., 1983, 129, 2621-2628.)
Table 7
Calculated pKa and pKint for Some Titratable Residues from the Active Site of S. aureus PC1 Wild Type (3blm) and Lys73His Mutant
(1dja) Compared with E. coli TEM-1 (1btl).
pKa (pKint)
Enzyme
εin
pI
SA PC1
SA PC1
20
4
10.0
9.52
SA PC1 K73H 20
SA PC1 K73H 4
ND
ND
24.3 (14.8) 17.9 (10.7)
1.01 (4.50)
21.5 (18.9) >30 (9.96)
-1.32 (6.92)
His-73 (H; H )
25.3 (16.3) 6.87 (6.03; 6.05) 0.614 (6.14)
>30 (23.4) 8.01 (2.08; 3.47) -3.34 (13.2)
5.06
5.09
Lys-73(C)
24.9 (15.0) 16.6 (10.4)
21.2 (19.4) >30 (7.99)
TEM-1
TEM-1
20
4
Ser-70 (A)
Lys-73(C)
Glu-166 (A) Lys-234 (C) Arg-244 (C) Asp-214 (A) Asp-233 (A)
2.85 (4.80)
2.82 (8.07)
14.2 (10.5)
13.6 (916)
>30 (19.1)
>30 (45.3)
Asn-214
Asn-214
0.027 (4.66)
-3.22 (9.62)
17.2 (12.4)
26.4 (15.9)
17.4 (12.5)
16.9 (10.5)
Asn-214
Asn-214
1.07 (5.23)
~-1 (11.4)
13.3 (10.4)
17.3 (8.34)
28.2 (19.4)
>30 (47.2)
3.32 (5.00) 0.445 (4.46)
17.3 (11.3) -4.02 (9.01)
S. aureus PC1 pI is 10.1 (Zygmunt, D. J.; Stratton, C. W.; Kernodle, D. S. Antimicrob. Agents. Chemother. 1992, 36, 440-445.)
Table 8
Calculated pKa and pKint for Some Titratable Residues from the Active Site of the Model of the Michaelis Complex of E. coli TEM-1,
B. licheniformis 749/C Wild Type, its Glu166Ala Mutant, S. aureus PC1 Wild Type and its Lys73His Mutant with Penicillin G.
pKa (pKint)
Enzyme
εin Ser-70 (A)
Lys-73(C)
Glu-166 (A)
Lys-234 (C) Arg-244 (C) Asp-214 (A) Asp-233 (A)
12.5 (6.91) 13.9 (9.15) 2.05 (3.07) -1.57 (2.57)
3.80 (-6.19) 10.7 (-.35) >17 (5.12) -9.95 (2.03)
TEM-1 (91)
TEM-1 (91)
20 27.9 (12.4) 16.1 (7.02)
4 >28 (13.2) >30 (-5.56)
-0.662 (2.72)
-8.21 (1.96)
TEM-1
TEM-1
20 29.5 (15.4) 17.9 (9.96)
4 >30 (25.0) >30 (6.35)
0.785 (4.15)
-1.72 (6.88)
18.5 (10.9)
>30 (10.2)
23.5 (16.1) 4.93 (5.40) 0.213 (4.59)
>30 (29.4) 16.3 (13.2) -4.34 (9.54)
BCL w.t.
BCL w.t.
20 >30 (15.5) 19.4 (10.6)
4 >30 (25.0) >30 (9.19)
0.669 (4.07)
-2.95 (6.23)
19.5 (10.7)
>30 (9.34)
19.6 (11.7) Asn-214
26.6 (8.60) Asn-214
3.82 (3.48)
30 (8.74)
19.2 (11.8) Asn-214
26.6 (8.78) Asn-214
-2.52 (3.38)
30 (15.4) 19.8 (10.1)
4 >30 (24.4) >30 (6.70)
His -73 (H; H)
20 >30 (16.7) 5.56 (4.39; 5.02)
4 >30 (29.2) 30 (14.2) -5.64 (0.607) 23.0 (12.1) 6.68 (3.15)
Intact
Yes
20 8.45 CHARMM22
26.0 (14.5) 15.4 (9.70) 7.76 (12.1) 21.7 (10.7)
Intact
Yes
4 7.77 CHARMM22
>20 (17.1) -1.49 (5.18) >18 (18.4) >14 (10.2)
Intact
No
20 6.46 AMBER91
26.5 (12.3) 14.3 (7.24) 5.23 (9.01) 12.2 (7.18)
Intact
No
4 4.88 AMBER91
>30 (12.8) -5.93 (-1.02) 21.2 (10.3) 4.17 (.811)
Intact
No
20 8.49 AMBER94
25.8 (14.4) 15.0 (9.40) 8.12 (11.5) 19.6 (10.5)
Intact
No
4 7.60 AMBER94
>30 (16.9) -3.52 (3.96) 12.9 (16.2) >30 (9.43)
Intact
No
20 8.45 CHARMM22
26.5 (14.5) 15.1 (9.51) 7.76 (11.6) 20.3 (10.5)
Intact
No
4 7.69 CHARMM22 ≈26.3 (17.1) -2.76 (4.32) 14.7 (16.7) >30 (9.57)
Intact
No
20 8.63
PARSE3
23.1 (14.0) 12.4 (9.66) 8.36 (11.4) 19.8 (10.7)
Intact
No
4 7.64
PARSE3
23.5 (15.5) -1.01 (5.51) 13.6 (16.1) >30 (10.6)
Michaelis
No
20 ND
AMBER91
>30 (13.8) 18.8 (8.68) 10.4 (9.52) 25.0 (7.56)
Michaelis
No
4 ND
AMBER91
>30 (19.7) 4.97 (2.17) >30 (11.0)
Michaelis
No
20 ND
AMBER94
>30 (16.2) 20.6 (10.7) 12.2 (12.0) 26.8 (10.6)
Michaelis
No
4 ND
AMBER94
>30 (28.4) 12.2 (9.95) 19.1 (19.5) >30 (9.71)
≈30 (-1.38)
E. cloacae P99 pI = 8.2; 7.8; 8.3; 8.25; 8.95 (Goering, R. V.; Sanders, C. C.; Sanders, W. E., Jr.;
Guay, R.; Guerin, S. Rev. Infect. Dis. 1988, 10, 786-792; Goward, C. R.; Stevens, G. B.;
Hammond, P. M.; Scawen, M. D. Chromatogr. 1988, 457, 317-324; Minami, S.; Inoue, M.;
Mitsuhashi, S. Antimicrob. Agents. Chemother. 1980, 18, 853-857; Seeberg, A. H.; TolxdorffNeutzling, R. M.; Wiedemann, B. Antimicrob. Agents. Chemother. 1983, 23, 918-925.)
Calculated sequence-based pI is 8.24 for positions 22-380 as in x-ray sequence.
Table 10
Calculated pKa and pKint for Some Titratable Residues from the Active Site of the DDTranspeptidase R61 Streptomyces sp. (1cef) and a Model of its Michaelis Complex with
Penicillin G.
Enzyme His εin pI
tautomerism
Parameter
Set
pKa (pKint)
Ser-62 (A)
Lys-65 (C)
Tyr-159 (A)
18.1 (8.38)
7.69 (10.2)
-1.37 (3.79)
27.0
9.21
7.84
9.31
>24
9.23
7.48
9.20
6.92
30
>30
>30
28.3
18.5
≈30
27.9
>30
29.8
(14.0) -1.97 (1.71)
(14.5) 23.6 (10.3)
(17.0) 13.1 (7.98)
(14.1) 21.9 (10.3)
(14.7) 8.55 (8.45)
(14.3) 21.8 (10.1)
(16.2) 12.9 (7.27)
(16.4) 20.5 (12.6)
(21.3) ~10.5 (15.7)
Michaelis
Michaelis
Michaelis
Michaelis
Michaelis
Michaelis
No
No
No
No
Yes
Yes
20
4
20
4
20
4
ND
ND
ND
ND
ND
ND
>30
>30
>30
>30
>30
>30
(13.2)
(16.1)
(14.8)
(21.0)
(17.8)
(33.1)
AMBER91
AMBER91
AMBER94
AMBER94
AMBER94
AMBER94
24.7
5.81
25.8
20.7
25.9
>18
(13.0)
(11.6)
(17.3)
(11.4)
(16.5)
(11.2)
(15.1)
(13.7)
(22.6)
His-298 (C)
(-3.44)
(6.39)
(6.92)
(6.24)
(5.43)
(5.78)
(4.25)
(7.94;7.26)
(9.72; 8.22)
(8.30) 14.1 (9.82) -3.58 (1.36)
(-0.21) >30 (12.6) 30 (17.3) 30 (28.4)