The functional role of cysteines in isopenicillin N synthase. Correlation of cysteine reactivities toward sulfhydryl reagents with kinetic properties of cysteine mutants.
Isopenicillin N synthase (IPNS) from Cephalosporium acremonium contains 2 cysteine residues in positions 106 and 255 which are invariant in all IPNS sequences reported to date (Miller, J.R., and Ingolia, T.D. (1989) Mol. Microbiol. 3, 689-695). Although these residues have been postulated to play a role in catalysis (Samson, S.M., Chapman, J.L., Belagaje, R., Queener, S., and Ingolia, T.D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5705-5709) as well as enzyme inactivation (Perry, D., Abraham, E.P., and Baldwin, J.E. (1988) Biochem. J. 255, 345-351) little information exists regarding their oxidation state and reactivity. In this paper, the functions of these cysteines have been addressed by chemical modification techniques in combination with site-directed mutagenesis. In the intact wild type protein, both cysteines are inert toward 5,5'-dithiobis-(2-nitrobenzoic acid) and iodoacetic acid. However, Cys-106, but not Cys-255, can be slowly modified by N-ethylmaleimide, and its modification is partially blocked by the presence of a substrate analog inhibitor. Complete modification of both cysteines by sulfhydryl reagents requires unfolding of the protein but not the presence of a disulfide reducing agent. The thiol content of IPNS is shown to be the same before and after exposing the enzyme to substrate even though during catalysis the enzyme is rapidly inactivated. The impact on catalysis due to alteration of the cysteines has been assessed using three site-specific mutants: Cys-106----Ser, Cys-255----Ser, and Cys-106,255----Ser. These mutant proteins have been purified as apoenzymes with the nature of the mutation verified by peptide mapping. The stoichiometry of metal required for activity remains as one equivalent of Fe2+/mol of enzyme in the mutants as in wild type IPNS. Compared with wild type, Cys-255----Ser shows a reduction in Vmax by 33%, and an increase in Km by 1.4-fold, while Cys-106----Ser and Cys-106,255----Ser, which have identical kinetic properties, exhibit a decrease in Vmax by 63% but an elevation of Km by 14-fold. The data presented demonstrate that 1) both cysteines are free thiols; 2) Cys-106 is more exposed than Cys-255; 3) substrate-induced inactivation is not caused by cysteine modification; 4) neither cysteine is absolutely essential for bond making or breaking events during catalysis.(ABSTRACT TRUNCATED AT 400 WORDS)
Kriauciunas
A
A
Department of Biochemistry, Lilly Research Laboratories, Indianapolis, Indiana 46285.
Frolik
C A
CA
Hassell
T C
TC
Skatrud
P L
PL
Johnson
M G
MG
Holbrook
N L
NL
Chen
V J
VJ
D000164:Acremonium / Q000201:enzymology; D000595:Amino Acid Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014357:Trypsin
D013438:Sulfhydryl Compounds; D010088:Oxidoreductases; C038370:isopenicillin N synthetase; D014357:Trypsin; D003545:Cysteine; D005033:Ethylmaleimide