Characterization of Manganese(II) Binding Site Mutants of Manganese Peroxidase

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• 作者：Katsuyuki Kishi ; Margo Kusters-van Someren ; Mary B. Mayfield ; Jie Sun ; Thomas M. Loehr ; and Michael H. Gold
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：July 9, 1996
• 年：1996
• 卷：35
• 期：27
• 页码：8986 - 8994
• 全文大小：444K
• 年卷期：v.35,no.27(July 9, 1996)
• ISSN：1520-4995

文摘

A series of site-directed mutants, E35Q, E39Q, andE35Q-D179N, in the gene encodingmanganese peroxidase isozyme 1 (mnp1) fromPhanerochaete chrysosporium, was created byoverlapextension, using the polymerase chain reaction. The mutant geneswere expressed in P. chrysosporiumduring primary metabolic growth under the control of theglyceraldehyde-3-phosphate dehydrogenasepromoter. The mutant manganese peroxidases (MnPs) were purifiedand characterized. The molecularmasses of the mutant proteins, as well as UV-vis spectral features oftheir oxidized states, were verysimilar to those of the wild-type enzyme. Resonance Raman spectralresults indicated that the hemeenvironment of the mutant MnP proteins also was similar to that of thewild-type protein. Steady-statekinetic analyses of the E35Q and E39Q mutant MnPs yieldedK_m values for the substrate Mn^IIthat were~50-fold greater than the corresponding K_mvalue for the wild-type enzyme. Likewise, thek_cat values forMn^II oxidation were ~300-fold lower than that forwild-type MnP. With the E35Q-D179N double mutant,the K_m value for Mn^II was~120-fold greater, and the k_cat value was~1000-fold less than that for thewild-type MnP1. Transient-state kinetic analysis of the reductionof MnP compound II by Mn^II allowedthe determination of the equilibrium dissociation constants(K_D) and first-order rate constants forthemutant proteins. The K_D values wereapproximately 100-fold higher for the single mutants andapproximately 200-fold higher for the double mutant, as compared withthe wild-type enzyme. The first-order rate constants for the single and double mutants were ~200-foldand ~4000-fold less, respectively,than that of the wild-type enzyme. In contrast, theK_m values for H₂O₂ andthe rates of compound Iformation were similar for the mutant and wild-type MnPs. Thesecond-order rate constants for p-cresoland ferrocyanide reduction of the mutant compounds II also were similarto those of the wild-type enzyme.