[591

TOYOCAMYCIN NITRILE HYDROLASE

759

PURIFICATION OF CLINDAMYCIN PHOSPHOTRANSFERASE

Volume Protein (ml) (mg/ml)

Enzyme fraction Crude lysate Manganous chloride -F ammonium sulfate DEAE-cellulose

200 10

6.5 31

5

0.18

Total activity (units X 10-3)

Specific activity Fold (X 10-3) purified

180 114

0.13 0.36

6.2

6.90

1 2.7

Yield (%) 100 63

53

3.4

maintains activity at 25 ° and 30 ° for at least 1 hr. I t displays greater initial activity at 37 ° than at 30 ° but it is inactivated readily at t h a t temperature. Stoichiometry. Although clindamycin A T P stoichiometry would be expected to be mole for mole in the reaction, optimum phosphorylation is obtained with a large excess of A T P over clindamycin. This m a y be explained by the fact t h a t the enzyme preparation still has marked adenosine triphosphatase activity after a m m o n i u m sulfate fractionation.

[59] T o y o c a m y c i n

Nitrile

Hydrolase

By T. UEMATSU and R. J. SUHADOLNIK NHz N/, ~ " ~ -

CN

NH2 N~"'~

I

C-NH, I

Ribo~e

TOYOCAMYCIN

0

Rlbose

SANGIVAMYCIN

Principle. The assay measures the conversion of toyocamycin to sangivamycin in the presence of t o y o c a m y c i n nitrile hydrolase. 1

Assay Reagents. Reaction mixture, 0.1 ml contains: Potassium phosphate buffer, 0.1 M, p H 6.5 [ G - 3 H ] T o y o c a m y c i n , 0.32 mM, 4 Ci/mole T o y o c a m y c i n nitrile hydrolase, 5.7 units 1T. Uematsu and R. J. Suhadolnik, Arch. Biochem. Biophys. 162, 614 (1974).

760

ANTIBIOTIC INACTIVATION AND MODIFICATION

[59]

Procedure. The reaction mixture is incubated at 30 ° for 30 min. Control assays are performed with boiled enzyme. The reactions are terminated by the addition of 0.1 ml ethanol-acetic acid (9:1, v/v) followed by heating at 90 ° in a steam bath for 10 min. The protein is removed by centrifugation. Carrier sangivamycin (30 ~g) is added to the supernatant. The mixture is spotted on a Whatman No. 3 MM paper and developed with water. The area corresponding to sangivamycin (RI 0.80) is cut out and eluted with 0.1 N HC1; an aliquot is counted by liquid scintillation counting. Definition o] Unit and Specific Activity. A unit of enzyme is defined as the formation of 1 nmole of sangivamycin per 30 min at 30 ° in 0.1 ml of incubation mixture. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Murphy and Kies. 2

Purification Procedure

Step 1. Preparation o] Cell-Free Extract. All purification steps are carried out at 0-4% The cells (Streptomyces rimosus ATCC 14673) are harvested by centrifugation at 4 °, 48 hr after inoculation. The cells then are washed twice with 0.15 M KC1, centrifuged, lyophilized, and stored at --20% The dried cells (5 g), obtained from 600 ml of medium, are suspended in 100 ml of 0.10 M phosphate buffer (pH 7.4) and disrupted with a French press (twice at 16,000 psi). The homogenate is suspended in 50 ml of buffer, stirred for 10 rain, and centrifuged. The sediment is washed with the buffer. The supernatant and washings are combined and dialyzed overnight with 2 liters of 1 mM EDTA (pH 7.4) ; volume 97 ml, 1014 mg protein. Step 2. Ammonium Sul]ate Fractionation. The crude extract (97 ml, 1014 mg protein) is brought to 30% saturation by the :gradual addition of solid ammonium sulfate over a 30-min period with stirring. The precipitate is removed by centrifugation (35,000 g, 10 min). Ammonium sulfate is added to the supernatant to 55% saturation over a 10-rain period, and centrifuged for 10 rain (15,000 g). The precipitate is dissolved in 10 ml of water, and dialyzed overnight with 2 liters of 1 mM EDTA (pH 7.4) ; volume 15 ml, 201 mg protein. Step 3. Protamine Sul]ate Treatment. A 1% protamine sulfate solution (pH 6.8) is added to 15 ml (201 mg protein) until the A2so/A26o of the supernatant becomes 0.90. The solution is centrifuged at 20,000 g, for 10 min. The pellet is suspended in 5 ml of 0.5 M phosphate buffer (pH 6.5). After stirring for 2 hr, the suspension is centrifuged at 20,000 g 2j. B. Murphy and M. W. Kies, Biochim. Biophys. Acta 45, 382 (1960).

[59]

TOYOCAMYCIN

NITRILE

HYDROLASE

©

Z

©

~v

Z

.o

©

761

762

ANTIBIOTIC INACTIVATION AND MODIFICATION

[59]

for 10 min. The pellet is discarded and the supernatant is dialyzed against 3 liters of 1 mM EDTA, 3.5 hr; volume 8.5 ml, 28 mg protein. Step 4. Hydroxyapatite Adsorption. The supernatant (8.5 ml; 28 mg protein) is passed through a hydroxyapatite column (10 ml). The elution of protein is carried out stepwise with 10 ml of 0.05, 0.08, 0.10, 0.12, 0.16, and 0.20 M phosphate buffer (pH 6.8). The hydrolase is eluted with 0.12 M buffer; volume 13.5 ml, 2.2 mg protein. The overall purification is shown in the table.

Properties Specificity. The purified enzyme would only use toyocamycin as the substrate.

Coenzyme, Metal Requirements, and Activators. The enzyme does not require coenzyme, metal ions, nor activators for activity. pH Optimum. The enzyme has an optimum pH at 6.5. Nonenzymic conversion of toyocamycin to sangivamycin occurs only when the pH is more alkaline than 10. Ej~ect o] Metal Ions. Zn 2÷, Mg 2+, Cu ~÷, Fe 2+, and Hg 2+ (all at 2.5 mM) inhibit the enzyme (47, 66, 100, 100 and 100%, respectively). Inhibitors. The following compounds (10-60 mM) inhibit the hydrolase: p-hydroxybenzonitrile, tubercidin, ricinine, tubercidin 5'monophosphate, nicotinonitrile, hydroxytoyocamycin, demethylricinine, O,N-didemethylricinine. The percent inhibition is between 81 and 82%. p-Hydroxybenzonitrile and tubercidin are competitive inhibitors of the hydrolase; K~ = 5.7 and 8.0 mM, respectively. Michaelis Constant. The Km for toyocamycin is 0.5 mM. The curves are linear. At 1.7 mM toyocamycin and 5.3 units of protein, product formation is linear for 60 min at 30°; when the protein is varied between 43 and 172 ~g, there is a linear increase in product formation. Ef]ect of Heat. When the enzyme is heated for 10 rain at 40 ° (0.4 mg protein, 0.6 mM [G-3H]toyocamycin), there is a 30% loss of activity; heating for 10 min at 60 ° results in complete loss of enzyme activity.