The Enzymes

Sumner, James B.; Myrbäck, Karl

doi:10.1097/00010694-195107000-00008

“…In general, however, it is a concentration just a few percent less than that which yields an amorphous precipitate (Sumner & Somers, 1943), and this can be determined for a macromolecule under a given set of conditions using only minute amounts of material (McPherson, 1982). To determine the approximate insolubility points with a particular precipitant, a 10 ml droplet of a 5-15 mg ml This diagram, based on an analysis of nearly 2800 examples, shows the distribution of the number of protein crystals grown as a function of pH.…”

Section: Figurementioning

confidence: 99%

Introduction to protein crystallization

McPherson

¹

,

Gavira

²

2013

Acta Crystallogr F Struct Biol Commun

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Protein crystallization was discovered by chance about 150 years ago and was developed in the late 19th century as a powerful purification tool and as a demonstration of chemical purity. The crystallization of proteins, nucleic acids and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by the manipulation of various parameters that include temperature, ionic strength and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch and liquid-liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years owing to the advent of practical, easy-to-use screening kits and the application of laboratory robotics. A brief review will be given here of the most popular methods, some guiding principles and an overview of current technologies.

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“…In general, however, it is a concentration just a few percent less than that which yields an amorphous precipitate (Sumner & Somers, 1943), and this can be determined for a macromolecule under a given set of conditions using only minute amounts of material (McPherson, 1982). To determine the approximate insolubility points with a particular precipitant, a 10 ml droplet of a 5-15 mg ml À1 protein solution can be placed in the well of a depression slide and IYCr crystallization series This diagram, based on an analysis of nearly 2800 examples, shows the distribution of the number of protein crystals grown as a function of pH.…”

Section: Figurementioning

confidence: 99%

Introduction to protein crystallization

McPherson

¹

2004

Methods

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Protein crystallization was discovered by chance about 150 years ago and was developed in the late 19th century as a powerful purification tool and as a demonstration of chemical purity. The crystallization of proteins, nucleic acids and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by the manipulation of various parameters that include temperature, ionic strength and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch and liquid-liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years owing to the advent of practical, easy-to-use screening kits and the application of laboratory robotics. A brief review will be given here of the most popular methods, some guiding principles and an overview of current technologies.

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“…Spectrophotometric studies showed that the nickel ion has an essential role in catalysis and that the substrate and inhibitors of urease bind to the nickel ion [13, 141. A model for the mechanism of action of urease, taking into consideration the nickel ion, has been proposed [15].The presence of sulfhydryl groups in a protein was first described for jack bean urease [16]. The sulfhydryl groups in the enzyme are classified into the exposed ones which are titrated in the native conformation and the buried ones which can be titrated only in the presence of denaturant.…”

mentioning

confidence: 99%

“…The presence of sulfhydryl groups in a protein was first described for jack bean urease [16]. The sulfhydryl groups in the enzyme are classified into the exposed ones which are titrated in the native conformation and the buried ones which can be titrated only in the presence of denaturant.…”

mentioning

confidence: 99%

The structure of jack bean urease

Takishima

¹

,

Suga

²

,

Mamiya

³

1988

European Journal of Biochemistry

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The amino acid sequence of jack bean urease has been determined. The protein consists of a single kind of polypeptide chain containing 840 amino acid residues. The subunit relative molecular mass calculated from the sequence is 90770, indicating that urease is composed of six subunits. Out of 25 histidine residues in urease, 13 were crowded in the region between residues 479 and 607, suggesting that this region may contain the nickelbinding site. Limited tryptic digestion cleaved urease at two sites, Lys-128 and Lys-662. Proteolytic products were not dissociated and retained full enzymatic activity. Five tryptic peptides containing the reactive cysteine residues were isolated and characterized with the aid of sulfhydryl-specific reagents, N-iodoacetyl-N'-(5-sulfo-lnaphthy1)ethylenediamine and N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide. The reactive cysteine residues were located at positions 59,207, 592,663, and 824. The possibility that Cys-59, Cys-207, Cys-663, and Cys-824 are involved in the urease activity of the enzyme has been eliminated. Cys-592, which is essential for enzymatic activity, is located in the above-mentioned histidine-rich region.Jack bean urease was the first enzyme isolated as a crystalline protein by Sumner in 1926 [l]. However, the subunit structure of this enzyme has been somewhat ambiguous. The relative molecular mass reported first for the native urease was 480000 [2] while recently the value of 590 000 was reported [3]. The values reported for the monomer range over 30000-97000 [3-61. On the other hand, the subunits of ureases from microorganisms appear to be smaller than jack bean urease in size and number [7-91. Recently it was suggested that one large and two small polypeptides may be generally associated with microbial ureases [lo].Jack bean urease is also the first example of a nickel metalloenzyme and contains two nickel ions per 96600-M, subunit [ l l , 121. Spectrophotometric studies showed that the nickel ion has an essential role in catalysis and that the substrate and inhibitors of urease bind to the nickel ion [13, 141. A model for the mechanism of action of urease, taking into consideration the nickel ion, has been proposed [15].The presence of sulfhydryl groups in a protein was first described for jack bean urease [16]. The sulfhydryl groups in the enzyme are classified into the exposed ones which are titrated in the native conformation and the buried ones which can be titrated only in the presence of denaturant. Urease has been reported to have 27 -35 reactive exposed cysteine residues per urease molecule [17 -191. These cysteine residues were divided into the inessential ones, which readily react with sulfhydryl reagents like 5,5'-dithiobis(2-nitrobenzoic acid) without activity loss, and the essential ones, which react more

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The Enzymes

Cited by 30 publications

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Introduction to protein crystallization

Introduction to protein crystallization

Introduction to protein crystallization

The structure of jack bean urease

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