Protein engineering of nitrile hydratase activity of papain: molecular dynamics study of a mutant and wild-type enzyme.

نویسندگان

  • Swarnalatha Y Reddy
  • Kalju Kahn
  • Ya-Jun Zheng
  • Thomas C Bruice
چکیده

The mechanism of hydrolysis of the nitrile (N-acetyl-phenylalanyl-2-amino-propionitrile, I) catalyzed by Gln19Glu mutant of papain has been studied by nanosecond molecular dynamics (MD) simulations. MD simulations of the complex of mutant enzyme with I and of mutant enzyme covalently attached to both neutral (II) and protonated (III) thioimidate intermediates were performed. An MD simulation with the wild-type enzyme.I complex was undertaken as a reference. The ion pair between protonated His159 and thiolate of Cys25 is coplanar, and the hydrogen bonding interaction S(-)(25).HD1-ND1(159) is observed throughout MD simulation of the mutant enzyme.I complex. Such a sustained hydrogen bond is absent in nitrile-bound wild-type papain due to the flexibility of the imidazole ring of His159. The nature of the residue at position 19 plays a critical role in the hydrolysis of the covalent thioimidate intermediate. When position 19 represents Glu, the imidazolium ion of His159-ND1(+).Cys25-S(-) ion pair is distant, on average, from the nitrile nitrogen of substrate I. Near attack conformers (NACs) have been identified in which His159-ImH(+) is positioned to initiate a general acid-catalyzed addition of Cys-S(-) to nitrile. Though Glu19-CO(2)H is distant from nitrile nitrogen in the mutant.I structure, MD simulations of the mutant.II covalent adduct finds Glu19-CO(2)H hydrogen bonded to the thioimide nitrogen of II. This hydrogen bonded species is much less stable than the hydrogen bonded Glu19-CO(2)(-) with mutant-bound protonated thioimidate (III). This observation supports Glu19-CO(2)H general acid catalysis of the formation of mutant.III. This is the commitment step in the Gln19Glu mutant catalysis of nitrile hydrolysis.

برای دانلود رایگان متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

QSARS OF ANTI-FUNGAL ACTIVITY OF FURAN CARBOXANILIDE DERIVATIVES AGAINST WILD AND MUTANT STRAINS OF USTILAGO MAYDIS

The structural requirements for the inhibitor activity of various furan carboxanilide derivatives against succinate dehydrogenase complex (SDC) activity in mitochondria of either wild or mutant strains of Ustilago maydis were investigated with the aid of Hansch QSAR analysis. It has been found that the inhibitor activity against both types of enzymes is best related to the ??? or ??M of th...

متن کامل

Purification and characterization of nitrile hydratase of mutant 4D of Rhodococcus rhodochrous PA-34

Nitrile hydratase (NHase; E.C. 4.2.1.84) has been purified and characterized using ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography from the mutant 4D of Rhodococcus rhodochrous PA-34. The SDS-PAGE and MALDI-TOF analysis of the purified enzyme revealed that it is dimmer consisting of α- and β-subunits with a molecular mass of 25 and 30 kDa, respectiv...

متن کامل

Effects of Condition Enzymatic Hydrolysis on Degrees of Hydrolysis and Antioxidant Activity of Head Protein of Bighead (Aristichthys nobilis)

Background and Objectives: Enzymatic hydrolysis is an effective practical technology that recovers valuable proteins from far-less farmed fish without losing their nutritional characteristics. Enzymatic hydrolysis of protein sources such as aquatic animals is a type of protein recycling. In the present study, effects of temperature, time and concentration of papain enzyme on hydrolysis degree a...

متن کامل

Molecular Docking and In Silico Study of Denileukin Diftitox: Comparison of Wild Type With C519S-Mutant

Background: Denileukin diftitox (trade name, Ontak) is the first recombinant immunotoxin (IM), in which the binding domain of diphtheria toxin has been replaced by the amino acid sequence of human interleukin-2 (DT389IL-2) using genetic engineering. Purity, stability, and structural property of the protein are critical factors for the scale-up production of this fusion protein. In this IM, loca...

متن کامل

Molecular dynamics simulations of the photoactive protein nitrile hydratase.

Nitrile hydratase (NHase) is an enzyme used in the industrial biotechnological production of acrylamide. The active site, which contains nonheme iron or noncorrin cobalt, is buried in the protein core at the interface of two domains, alpha and beta. Hydrogen bonds between betaArg-56 and alphaCys-114 sulfenic acid (alphaCEA114) are important to maintain the enzymatic activity. The enzyme may be ...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

عنوان ژورنال:
  • Journal of the American Chemical Society

دوره 124 44  شماره 

صفحات  -

تاریخ انتشار 2002